Abstract: In order to study the genetic diversity, genetic structure and maternal origin of some equine populations in Gansu Province, cytochrome b（Cytb） gene of mitochondrial DNA（mtDNA） was amplified by PCR from 133 individuals from four populations of horses in Gansu Province（49 Chakouyi horses, 20 Hequ horses, 30 Shandan horses and 34 Sunan horses） by DNA sequencing. The sequence characteristics, genetic diversity, genetic distance, genetic differentiation and variation of Cytb gene in four equine populations were analyzed, and phylogenetic trees and haplotype network relationship diagram were constructed by combining the Cytb gene sequence of other horse populations. The results showed that the total length of Cytb gene sequence in four equine populations was 1 140 bp, and the A+T content（54.6%） was greater than G+C content（45.4%）. A total of 46 polymorphic loci and 33 haplotypes were detected. The total haplotype diversity was 0.933 2±0.010 0; the total nucleotide diversity was 0.003 85±0.000 17, and the total mean nucleotide difference was 4.371 3. The average Tajima’s D value and Fu’s Fs value were-1.035 2 and-13.057, respectively. The ranges of genetic distance, genetic coefficient of variation and gene flow were 0.003 5-0.004 2, 0.019 23-0.091 32, 4.975-25.504, respectively. The genetic variation in the four herds（94.54%） was much higher than that among the populations（5.46%）. The 33 haplotypes from four equine populations were dispersed in six clades（A-F）. These results indicated that the four horse populations in Gansu Province were closely related, with high genetic diversity and multi-matrilineal origin.