In this study, we investigated polymorphic distributions of allelic frequencies and forensic genetic parameters of 21 novel autosomal microsatellite loci from 110 unrelated healthy individuals of Chinese Yi ethnic group. Expected heterozygosity, power of discrimination, and polymorphic information content ranged from 0.617 to 0.812, 0.777 to 0.936 and 0.560 to 0.790. The microsatellite loci showed high forensic efficiency. The total discrimination power and cumulate probability of exclusion were 0.99999999999999999986902 and 0.999998818, respectively. Locus-by-locus allelic frequencies were compared using analysis of molecular variance (AMOVA) method, and the statistically significant differences were observed between Yi group and Russian, Tujia, Kazak, Bai, Ningxia Han, Salar, Tibetan, and Uigur groups at 5, 6, 7, 7, 7, 8, 12, and 13 loci, respectively. The results of genetic distance comparisons, genetic structure analyses, and principal component analysis all indicated that the Yi group showed relatively short genetic relationships with Russian, Salar, and Bai group. The experimental results showed that the 21 loci in the multiplex system provided highly polymorphic information and forensic efficiency for forensic individual identification and paternity testing, also basic population data for population genetics and anthropological research.