Painting in the Low Countries in the 16th century was as rich as it was varied. A number of trends were continued and new genres appeared. The museum's exceptional collections take these different movements into account, just as it does the originality of the artists who created them.

The route opens with the Bruges and Antwerp schools of the beginning of the century, represented by key works by Gerard David, Quentin Metsys and Joos van Cleve. Then come the Antwerp mannerists, the Romanists, Jan Gossaert - who was the first artist to paint mythological nudes in the Netherlands, as demonstrated by Venus and Cupid - and Bernard van Orley, whose Triptych of the Virtue of Patience, produced for the regent Margaret of Austria, is one of his masterpieces. The beginning of the independent landscape is marked by the works of Joachim Patenier and Herri met de Bles. Jan van Hemessen, Pieter Aertsen, Joachim Beuckelaer and his brother, the presumed Huybrecht Beuckeleer, all illustrate the rise of genre painting.

This section also includes a major group of paintings by Pieter Bruegel the Elder, including the famous Census at Bethlehem. Other creations by the master are also represented by the copies painted by his son, Pieter the Younger, including the imposing Fight between Carnival and Lent, which was acquired in 1999. Among its 16th century Italian paintings, the museum has a number of fine Tintorettos, while Lucas Cranach the Elder and Conrad Faber evoke features of the German school.