| 1. Infant of Portugal Afonso, b. 24 Feb 1526 d. in infancy |
+ | 2. Infanta of Portugal Maria, b. 15 Oct 1527, Coimbra d. 12 Jul 1545, Valladolid (Age 17 years) |
| 3. Infanta of Portugal Isabella, b. 28 Apr 1529 d. in infancy |
| 4. Infanta of Portugal Beatriz, b. 15 Feb 1530 d. in infancy |
| 5. Infant of Portugal Manuel, b. 1 Nov 1531 d. 14 Apr 1537 (Age 5 years) |
| 6. of Portugal, Felipe Infant, b. 25 May 1533 d. 29 Apr 1539 (Age 5 years) |
| 7. Infant of Portugal Dinis, b. 26 Apr 1535 d. 1 Jan 1537 (Age 1 year) |
+ | 8. Crown Prince of Portugal Joao, b. 3 Jun 1537, Evora d. 2 Jan 1554, Lisboa (Age 16 years) |
| 9. Infant of Portugal Antonio, b. 9 Mar 1539 d. 20 Jan 1540 (Age 0 years) |