Genetic polymorphism and structure analysis of 29 Y-STR loci from populations of Hui, Miao and Yi in Guizhou area were studied, and their genetic relationships with seven other populations were analyzed. DNATyperTMY29 kit was used to detect samples in 309 individuals of Miao, 291 individuals of Hui and 331 individuals of Yi of Guizhou. The allele frequency and gene diversity of 29 Y-STR loci were analyzed, analysis of molecular variance (AMOVA) was analyzed by YHRD online software. 816 different haplotypes were observed in 931 unrelated individuals from the three ethnic groups of Hui, Miao and Yi in Guizhou, and the haplotype diversities were determined as 0.995?5, 0.998?9 and 0.999?1. The genetic diversity (GD) in Hui, Miao and Yi were 0.289?9 to 0.874?7, 0.252?3 to 0.915?5 and 0.316?2 to 0.947?9, individually. AMOVA analysis showed that the genetic distance among 10 populations ranged from 0.007 8 to 0.138?9. The results showed that the 29 Y-STR loci were highly polymorphic in Hui, Miao and Yi populations in Guizhou. The data obtained in this study can provide basic data support for forensic science and population genetics research of the above population.