Wolfgang of Anhalt, called the Confessor, who becomes prince of Anhalt-Köthen in 1508, is the second ruler in the world to introduce the Reformation to his country.

He is a co-signer of the Augsburg Confession in 1530, and after the Battle of Mühlberg in 1547 is placed under Imperial ban and deprived of his lands by Emperor Charles V. After the peace of Passau in 1552 he buys back his principality, but as he is childless he surrenders it in 1562 to his kinsmen the princes of Anhalt-Dessau.

Ernest I, Prince of Anhalt-Dessau (d. 1516) leaves three sons, John V, George III, and Joachim, who rule their lands together for many years, and who favor the Lutheran doctrine, which thus becomes dominant in Anhalt.

About 1546, the three brothers divide their principality and establish the lines of Zerbst, Plötzkau and Dessau.

This division, however, is only temporary, as the acquisition of Köthen, and a series of deaths among the ruling princes, enables Joachim Ernest, a son of John V, to unite the whole of Anhalt under his rule in 1570.Joachim Ernest dies in 1586, and his five sons rule the land in common until 1603, when owing to the lack of primogeniture, Anhalt is again divided, and the lines of Dessau, Bernburg, Plötzkau, Zerbst and Köthen are re-founded.