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The Mestizos of Oaxaca resulted from the admixture of Zapotecan Natives with Spaniards and Africans. We selected 112 donors from Oaxaca and applied next-generation sequencing to characterize exon and intron variants in complete or extended HLA genes. Some alleles found, are unique to Mexican Natives and most likely will be absent in most major ethnicities, namely: Caucasians, Africans or Asians. Among these are HLA-A*68:03:01, HLA-A*68:05:01, HLA-C*03:04:01:02, HLA-C*15:09, HLA-C*3:05, HLA-C*03:06:01, HLAB*39:05:01, HLA-B*35:14:01, HLA-B*35:12:01, HLA-B*35:43:01, HLA-B*40:05, HLA-B:40:08, HLA-B*51:02:01, HLA-B*35:24:01 and HLA-B*39:08. HLA-DQA1*05:05:01:05 and some HLA-DRB1 alleles were only present in Amerindians/Mestizos. Three haplotypes are unique to Mexican Natives, five to Middle-Eastern and Sephardi-Jews. We detected a novel HLA-DQA1*04:01:01 exon 4 variant. Any novel allele may have been positively selected to enlarge the peptide-binding repertoire, and some, like HLA-B*39:02:02 and HLA-B*39:05:01 were found with unique haplotype associations, suggesting convergent evolution events and/or allele lineage diversification. The allele frequencies were fairly evenly distributed in most HLA loci with the exception of HLADPB1. The application of NGS in Oaxaca is novel and will lead to better use in the clinical setting. It offers deep knowledge on the population structure, origins, migration, and discovery of new alleles and haplotypes that other techniques did not achieve.

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