In the present study, the polymorphism of human leucocyte antigen class II genes was investigated by the sequence-based typing method in two Chinese populations: the Miaos (n = 85) from Guizhou province and the Yaos (n = 66) from Yunnan province. These two populations exhibited certain similarity in their allelic distributions. Among 24 DRB1 alleles detected, DRB1*150101, DRB1*140101, DRB1*160201 and DRB1*090102 in Miao and DRB1*120201, DRB1*140101, DRB1*150101 and DRB1*090102 in Yao were highly predominant. Sixteen DQB1 alleles in total were found in these two populations among which DQB1*050201, DQB1*060101/060103 and DQB1*030101/0309 in both Miao and Yao and DQB1*050301 in Yao were commonly observed. In the 13 DPB1 alleles detected, the most frequent allele was DPB1*0501 in Miao and Yao followed by DPB1*02 and DPB1*1301. Frequent comparisons with other Chinese populations suggested the southern Chinese feature for both the Miao and Yao nationalities.