Abstract: To explore the genetic diversity and origin of Gansu indigenous sheep breeds determined by mitochondrial DNA Dloop sequence, one pair of primers was designed to amplify the mtDNA Dloop sequence of sheep. By sequencing we got the basic sequencing data. Then the haplotype, genetic diversity, phylogenetics, and Network analysis were carried out. The results showed that 3 sequences were 573 bp length, 117 sequences are 648 bp length. 77 haplotypes are identified in 117 sequences. Through analysis, it is found that haplotype proportion, haplotype diversity, nucleotide diversity and average number of nucleotide difference of Lanzhou Fattailed sheep and Minxian Black Fur sheeps were very low, but it was very high in Mongolian sheep. The phylogenetic and network analysis demonstrated that Gansu 6 sheep breeds were divided into 3 clusters. So we concluded that 4 repeat units was the characteristic of Gansu sheep mtDNA Dloop sequence. The genetic diversity in Mongolian sheep is the highest, and it is the lowest in Lanzhou Fattailed sheep and Minxian Black Fur sheep. Phylogenetic and network analysis of the 77 haplotypes identified in Gansu 6 sheep breeds supported that there are 3 maternal origins in Gansu local sheep breeds.