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Year 1540 (MDXL) was a leap year starting on Thursday (link will display the full calendar) of the Julian calendar.
- July 7 – Coronado captures Hawikuh, at this time known as part of Cíbola, but fails to find the legendary gold.
- July 9 – King Henry VIII of England divorces Anne of Cleves, his fourth Queen consort.
- July 28 – One of the most important political figures of the reign of Henry VIII of England, Thomas Cromwell, is executed on order from the king, on charges of treason. Henry marries his fifth wife, Catherine Howard, on the same day.
- August 15 – In Peru, the Spanish captain Garcí Manuel de Carbajal founded the Villa Hermosa de Arequipa, and one year after, Charles V of Germany and I of Spain gave the valley a status of 'city' by Royal Decree.
- September – Gibraltar is sacked by the fleet of Barbary pirate Ali Hamet, a Sardinian renegade in the service of the Ottoman Empire, and many of its leading citizens are taken as captives to Peñón de Vélez de la Gomera in Morocco. This leads to construction of the defensive Charles V Wall, at this time known as the Muralla de San Benito.
- September 3 – Gelawdewos succeeds his father Lebna Dengel, as Emperor of Ethiopia.
- September 27 – The Society of Jesus (Jesuits) is approved by Pope Paul III, in his bull Regimini militantis Ecclesiae.
- October 1 – Battle of Alborán: A Habsburg Spanish fleet, under the command of Bernardino de Mendoza, destroys an Ottoman fleet, commanded by Ali Hamet, off Alborán Island in the Mediterranean.
- October 18 – An expedition led by Spanish conquistador Hernando de Soto destroys the fortified village of Mabila in modern-day Alabama, killing paramount chief Tuskaloosa.
- January 18 – Catherine, Duchess of Braganza, claimant to the Portuguese throne in 1580 (d. 1614)
- January 24 – Edmund Campion, English Jesuit and Roman Catholic martyr (d. 1581)
- January 28 – Ludolph van Ceulen, German mathematician (d. 1610)
- February 12 – Won Gyun, Korean general and admiral during the Joseon Dynasty (d. 1597)
- February 23 – Hedwig of Brandenburg, Duchess of Brunswick-Wolfenbüttel (d. 1602)
- February 25 – Henry Howard, 1st Earl of Northampton, English aristocrat and courtier (d. 1614)
- March 1 – Enrique de Guzmán, 2nd Count of Olivares, Spanish noble (d. 1607)
- March 17 – Bernhard VII, Prince of Anhalt-Zerbst, German prince of the House of Ascania (d. 1570)
- April 3 – Maria de' Medici, Italian noble (d. 1557)
- April 8 – Toyotomi Hidenaga, Japanese warlord (d. 1591)
- May 9 – Maharana Pratap, Indian warrior king (d. 1597)
- May 14
- May 22 – James, Duke of Rothesay, Scottish prince (d. 1541)
- May 31 – Henry Cheyne, 1st Baron Cheyne, English politician and baron (d. 1587)
- June 3 – Charles II, Archduke of Austria, regent of Inner Austria (d. 1590)
- June 9 – Shima Sakon, Japanese samurai (d. 1600)
- June 11 – Barnabe Googe, English poet (d. 1594)
- June 29 – Ana de Mendoza, Princess of Eboli, Spanish countess (d. 1592)
- June 30 – Countess Palatine Elisabeth of Simmern-Sponheim, Duchess of Saxony (d. 1594)
- July 7 – John Sigismund Zápolya, King of Hungary (d. 1571)
- July 11 – Adolf of Nassau, Count of Nassau, Dutch soldier (d. 1568)
- July 16 – Alfonso Carafa, Italian cardinal (d. 1565)
- July 19 – Ludowika Margaretha of Zweibrücken-Bitsch, spouse of Count Philip V of Hanau-Lichtenberg (d. 1569)
- August 4 – Sisto Fabri, Italian theologian (d. 1594)
- August 5 – Joseph Justus Scaliger, French Protestant scholar (d. 1609)
- August 25 – Lady Catherine Grey, English noblewoman, potential successor to the throne (d. 1568)
- September 5 – Magnus, Duke of Holstein, Prince of Denmark (d. 1583)
- September 9 – John VII, Count of Oldenburg (d. 1603)
- October 1 – Johann Jakob Grynaeus, Swiss Protestant clergyman (d. 1617)
- November 12 – Anna of Veldenz, Margrave of Baden (d. 1586)
- November 16 – Princess Cecilia of Sweden (d. 1627)
- December 8 – Giovanni Vincenzo Gonzaga, Italian Catholic cardinal (d. 1591)
- December 21 – Thomas Schweicker, German artist (d. 1602)
- December 28 – Charles I, Duke of Mecklenburg (d. 1610)
- December 31 – Silvio Antoniano, Italian Catholic cardinal (d. 1603)
- date unknown
- Andrea Andreani, Italian wood engraver (d. 1623)
- Francis Drake, English sea captain, privateer, navigator, slaver, pirate and politician (d. 1596)
- Christopher Hatton, English politician (d. 1591)
- George Hastings, 4th Earl of Huntingdon, English nobleman (d. 1604)
- Pierre Jeannin, French statesman (d. 1622)
- François Viète, French mathematician (d. 1603)
- Amago Yoshihisa, Japanese samurai and warlord (d. 1610)
- c. January – Elizabeth Blount, mistress of Henry VIII of England (b. 1502)
- January 27 – Angela Merici, Italian religious leader and saint (b. 1474)
- March 30 – Matthäus Lang von Wellenburg, German statesman and archbishop of Salzburg (b. 1469)
- April 21 – Afonso of Portugal, Catholic cardinal (b. 1509)
- May 6 – Juan Luís Vives, Spanish scholar (b. 1492)
- May 22 – Francesco Guicciardini, Italian statesman and historian (b. 1483)
- June 16 – Konrad von Thüngen, German noble (b. c. 1466)
- July 22 – John Zápolya, King of Hungary (b. c. 1490)
- July 28 – Thomas Cromwell, 1st Earl of Essex, English statesman (executed) (b. c.1485)
- July 30
- August 23 – Guillaume Budé, French scholar (b. 1467)
- August 24 – Girolamo Francesco Maria Mazzola (Parmigianino), Italian artist (b. 1503)
- August 28 – Federico II Gonzaga, Duke of Mantua (b. 1500)
- September 2 – Lebna Dengel, Emperor of Ethiopia (in battle) (b. 1501)
- September 16 – Enrique de Borja y Aragón, Spanish noble of the House of Borgia (b. 1518)
- September 20 – Infante Duarte, Duke of Guimarães, son of King Manuel I of Portugal (b. 1515)
- October 5 – Helius Eobanus Hessus, German Latin poet (b. 1488)
- date unknown – Francisco de Ulloa, Spanish explorer