The Columbian Exchange resulted in a widespread movement of humans, plants and animals between the Old and New Worlds. The late 15th to early 16th century transfer of cattle from the Iberian Peninsula and Canary Islands to the Caribbean laid the foundation for the development of American creole cattle (Bos taurus) breeds. Genetic analyses of modern cattle from the Americas reveal a mixed ancestry of European, African and Indian origins. Recent debate in the genetic literature centers on the ‘African’ haplogroup T1 and its subhaplogroups, alternatively tying their origins to the initial Spanish herds, and/or from subsequent movements of taurine cattle through the African slave trade. We examine this problem through ancient DNA analysis of early 16th century cattle bone from Sevilla la Nueva, the first Spanish colony in Jamaica. In spite of poor DNA preservation, both T3 and T1 haplogroups were identified in the cattle remains, confirming the presence of T1 in the earliest Spanish herds. The absence, however, of “African-derived American” haplotypes (AA/T1c1a1) in the Sevilla la Nueva sample, leaves open the origins of this sub-haplogroup in contemporary Caribbean cattle.