John V of Anhalt-Zerbst (Dessau, 4 September 1504[1] – Zerbst, 4 February 1551[2]), was a German prince of the House of Ascania and ruler of the principality of Anhalt-Dessau.

John was the second (but eldest surviving) son of Ernest I, Prince of Anhalt-Dessau, by his wife Margarete, daughter of Henry I, Duke of Münsterberg-Oels, and granddaughter of George of Poděbrady, King of Bohemia.

Upon the death of his father in 1516, John and his brothers George III and Joachim I inherited Anhalt-Dessau as co-rulers according to the family law of the House of Ascania.

Margarete's stepson, the new Duke of Pomerania, Philip I, had to pay her a lifetime annuity of 1200 guilders from his personal treasure in order to cover the cost of her dowry.

From her marriage to George I, Margarete had a posthumous daughter, Georgia, who followed her mother to Anhalt; however, was decided that when she reached her eighth birthday (in 1539) she must be returned to Pomerania under the custody of her half-brother Philip I.