The portrait was probably commissioned by Mary of Hungary, sister of Charles V as it appears in her inventories rather than those of the Emperor. After her death in 1558 it entered the collection of Philip II. On the death of Philip the painting was exhibited, together with Philip II offering the Infante don Fernando to the Heavens, also by Titian, in the Casa del Tesoro in the Alcázar in Madrid. Both were valued at 200 ducados, a higher value than that assigned to any other work.

Between approximately 1604 and 1626 the canvas hung in the palace at El Pardo, then returned to the Alcázar. As one of the masterpieces of the Spanish royal collection it was displayed in the prestigious setting of the Salón Nuevo or Salón de los espejos. In the 18th century the painting was housed in the Palacio Real Nuevo before entering the Museo del Prado in 1827.