The Spanish Colonial Manuscripts document over three centuries of history related to New Spain and its northern provinces in the Americas. The collection consists of Inquisition records, civil suits and trials, trials of enslaved and Indigenous persons, Cortés family suits and documents, civil and government matters, ecclesiastical matters, exploration and missionization, Indigenous relations/Nahuatl manuscripts, and royal correspondences. A key focus of the collection centers on Mexico City and records related to the Mexican Inquisition.

Inquisition materials document the administrative, legal, spiritual, and social experiences of people within the Spanish Atlantic world. Three major thematic areas within the Inquisition records include 1) solicitation cases and priestly misconduct, 2) enslaved persons tried for blasphemy, and 3) large-scale trials against those suspected of practicing Judaism during the 1640s. Civil and government matters contain letters, censuses, personal, property, and town titles, claims, genealogies, viceregal pronouncements, receipts, official appointments, land grants, wills, and disputes from the early-16th through the mid-19th centuries. A strength in this area includes legal documents related to Hernán and Martín Cortés title and land claims, as well as compensation disputes following conquest. Topics cover such issues as money and property disputes, debts and embezzlement charges, bigamy and blasphemy, and other misconduct. Ecclesiastical records address the reach and limits of church authority, religious history, letters, directives, and decrees.

Notable names within these collections include Diego Columbus, Hernando de Soto, Hernán Cortés, Ponce de León, the Cortés family, Phillip II, Bartolome de las Casas, Carlotta and Maximillian, Duarte de León Jaramillo and family (daughters Clara and Ana Nuñez).