Virginia was born after the formal resignation of her father of the government on behalf of her half-brother Francesco. Cosimo I contracted a morganatic marriage with Camilla Martelli on 29 March 1570 on the advice of Pope Pius V, and this allowed him to legitimize their daughter on the principle of "per subsequens". Since that time, she lived with her parents at the Villa di Castello during the summer and in Pisa in winter. Cosimo I's older children resented their father's second marriage, and after the death of the Grand Duke in 1574, they imprisoned Camilla in the Florentine convent of Murate.