We analyzed the HLA class II allele frequencies in 50 healthy unrelated Mayan individuals. The relationship with other worldwide populations was studied by using HLA data from 71 different populations. The most frequent alleles were HLA-DRB1*04, HLA-DRB1*01, HLA-DQB1*0302 and HLA-DQB1*0501. When comparisons with other Mexican Amerindian groups were made, some differences were observed. Mayans showed an increased frequency of HLA-DRB1*01 when compared to Nahuas, Mayos, Teenek and Mazatecans (p < 0.05), whereas the HLA-DRB1*04 was increased in Mayans when compared to Nahuas (p < 0.05). The analysis of HLA-DQB1 alleles showed an increased frequency of DQB1*0302 in Mayans when compared to Nahuas and Mazatecans (p < 0.05), whereas the frequency of HLA-DQB1*0301 was decreased in Mayans when compared to Nahuas, Mayos, Teenek and Mazatecans (p < 0.05). Decreased frequency of HLA-DQB1*0501 in Mayans when compared to Nahuas was found. Neighbour Joining dendrogram shows that Mexican Mayans are genetically close to some of the most ancient groups living in Mexico and some South American Amerindians. However, Guatemalan Mayans do not cluster together with Mexican Mayas showing that languages do not correlate with genes, particularly in Amerindians. The data corroborate the restricted polymorphism of HLA-DRB1 and DQB1 alleles and the high frequency of HLA-DRB1*04 and HLA-DQB1*0302 in Mayans from Mexico.