Charles V was in mourning and dressed in black for the rest of his reign following her death and did not remarry. He wanted a portrait of his deceased wife that was a likeness as he remembered her. For Titian, he had only the portraits painted by William (Gilliam) Scrots and Rubens for references to her as a likeness.

Two paintings were produced by Titian for Charles V. In the first painting, Isabella is seated and holding flowers on her lap with her left hand. She is dressed in black with the Imperial Crown at the right hand back wall. With the later painting, Isabella is sat in the same position wearing red and gold embroidered clothing and has a landscape behind her instead of the Crown. She is holding a book in her right hand, and the flowers have gone.

The first painting is very similar to the portrait of Isabella that probably was painted by William Scrots, and would have been a near likeness of her. The later painting has a much softer face and the nose has been straightened to the classical aquiline-shape nose. This change in appearance may have been Charles V's romantic memory of her.

The later painting, with Isabella wearing a red and gold dress, was the one Charles V used as the official portrait of his beloved wife, Isabella. This painting was part of the Royal Spanish Collection and is now in the Prado Museum in Madrid. Titian used oils on canvas and paid attention to the richly decorated gown with gold embroidery and red cloth. He added jewellery; a pearl necklace with an ornate clasp and a jewelled ring on the left hand.

He went into particular detail on the dress, with fine details, to give the impression of the imperial grandeur and great wealth she had as wife of the Holy Roman Emperor. The red and gold decoration is carried through with the wall covering, or curtain behind her. The window to the left of Isabella shows mountainous landscape, typical of the mountainous areas of her home. Leonardo da Vinci and Raphael were already painting portraits in this style with the subject near a window and Titian could well have been following this.

Charles V wanted to remember his wife as a pious and loving wife and mother. He gave her a softened look on her face and added a book to her right hand. She was the owner of a large library, mainly of pious and religious books, many of them she collected before she married Charles. Titian made sure she looked educated and well-read. She did make sure her children were well-educated, and she brought this love of books to her reign. Charles V wore black and never remarried after Isabella died. Isabella took charge of the empire when Charles was away, and Titian shows her as an ageless beauty, intelligent and with an air of subtle authority.