To find a rapid single nucleotide polymorphism (SNP) loci typing method in the mitochondrial DNA (mtDNA) coding regions, we genotyped 16 SNP loci in the mitochondrial DNA coding region in Han, Miao and Tujia populations by the multiplex-amplified product-length polymorphism (mAPLP) technique. This method generates allele-specific fragments that are different in length through PCR amplification using allele-specific forward (or reverse) primers different in size and a common reverse (or forward) primer. Results showed that both of the allelic frequency of 3970T in Han and Tujia populations were 17%, which were significantly different from that of in the Miao population (P0.01). The allelic frequency of 8020A in Han population was 6% , which was different from that of in the Miao and Tujia populations (P0.05) . In all of 300 samples, a total of 45 different haplotypes were identified, 12 of which were found in all the three populations, 10 were shared by two populations and 23 haplotypes existed only in one of the populations. Among these 23 haplotypes, 8, 6 and 12 haplotypes were exclusively observed in the Han, Miao and Tujia populations, respectively.