Bovine leukocyte antigens (BoLA) have been widely studied because of their primary function in the recognition of pathogens by the immune system. To date, however, the characterization of the BoLA-DRB3 gene in Latin American Zebu and mixed zebuine breeds is scarce. By a sequence-based typing method, here we sequenced exon 2 of BoLA class II DRB3 gene in 264 animals from the five most commonly used breeds in northern Argentina (Creole, Brahman, Braford, Brangus, and Nellore).The Bos taurus, Bos indicus, and mixed breeds analyzed here contained 61 previously reported alleles. Genetic diversity was high at both allelic and nucleotide sequence levels, particularly in the mixed breeds Braford and Brangus. In contrast to previous reports on DRB3 diversity, no evidence of balancing selection was found in our data. Differentiation among breeds was highly significant, as shown by FST (FST = 0.052, P < 0.001) and cluster analyses. In accordance with historical origin of the breeds, UPGMA trees and metric multidimensional scaling (MDS) analyses showed that Creole is distantly related to the other zebuine breeds. Among them, Brahman, Braford, and Brangus exhibited the closest affiliations. Despite the overall differentiation of the breeds, analysis of the peptide binding regions at the aminoacid level revealed that the key aminoacids involved in peptide recognition are greatly conserved suggesting little influence of domestication and breeding in functional MHC variability. In sum, this is the first report of BoLA-DRB3 diversity in pure and mixed Bos indicus cattle breeds from Argentina. Knowledge of BoLA-DRB3 variability in breeds adapted to tropical and subtropical environments contributes not only to the characterization of MHC diversity but also to the design of peptide-based vaccines.