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At the end of 1626, Maria Anna was betrothed to Ferdinand, the younger brother of her first fiancé, and the new heir of Emperor Ferdinand II. Ferdinand was her first cousin, being the son of her mother’s brother. The formal engagement was preceded by a series of negotiations which were conducted in 1625.

That same year, Prince Ferdinand was crowned King of Hungary, and in 1627 King of Bohemia. In the negotiations were included all the life aspects of the Infanta at the court of her future spouse. Despite the desire of the groom that Maria Anna’s confessor would be the Jesuit Ambrosio Penalosa, the appointment eventually went to Capuchin Diego Quiroga. In the marriage contract signed by both parties in 1628, it was noted that Maria Anna could retain her rights of inheritance over the Spanish throne, while her older sister Infanta Anna, married to King Louis XIII of France in 1615, was forced to renounce to her rights.

Maria Anna had left Madrid for Vienna in December 1629, fully three years after her engegement, and nearly five years after the proposal for marriage was first mooted. The journey, once embarked upon, took more than a year to complete. En route by sea, in Genoa complications arose due to an epidemic of the plague that erupted in the Italian Peninsula. For this reason, the party was unable to stop in Bologna, where Cardinal Antonio Barberini (nephew of Pope Urban VIII), was waiting for the Infanta to give her the Golden Rose.

The party moved to Naples, where Maria Anna finally received the award. Leaving the Kingdom of Naples, the Infanta crossed the Papal States, having made a pilgrimage to the Basilica della Santa Casa. On this section of her journey, Maria Anna was accompanied by Roman aristocracy, led by another nephew of Pope Urban VIII, Taddeo Barberini, Prince of Palestrina. On 26 January 1631, she arrived in Trieste, where she met Archduke Leopold Wilhelm of Austria, her future brother-in-law, who would first stand in for his brother at a wedding-by-proxy and then escort the Infanta to Vienna. That very day, Maria Anna was married to King Ferdinand of Hungria-Bohemia per procura with Archduke Leopold Wilhelm serving as the proxy.

Before the official wedding, King Ferdinand, not trusting the previous portraits that he had seen of the Infanta, decided to secretly view his bride. The Royal oberhofmeister asked for an audience with Maria Anna; on this visit, he was accompanied by some nobles, among whom was her groom. Struck by the beauty of the Infanta, King Ferdinand immediately revealed his identity and began a conversation with Maria Anna in Spanish. The love and respect that the future emperor felt for his wife lasted throughout their marriage. He was never unfaithful to her and never had any illegitimate children.

In Vienna on February 20, 1631, Maria Anna was married to King Ferdinand of Hungary-Bohemia. The festivities lasted a whole month. The marriage was described as friendly. Maria Anna was described as happy-tempered, friendly, and intelligent, and she was able to relieve the feelings of the rather melancholic Ferdinand.