Abstract: Binglangjiang buffalo is the first indigenous river type buffalo which has been found in western Yunnan province of China in recent years. In order to elucidate genetic background information on its genetic diversity, genetic composition, genetic relationship with typical river and swamp buffalo, and the gene introgression from swamp buffalo, 141 random samples from Binglangjiang buffalo population and 24 samples from control river buffalo population (Murrah) and 41 samples from control swamp buffalo population (Diandongnan buffalo) were genotyped by employing 30 microsatellite DNA markers. The result showed that a total of 253 alleles were identified in Binglangjiang buffalo (including 54 alleles shared among the three populations assayed, 58 alleles only shared between Binglangjiang and Murrah buffalo, 73 alleles only shared between Binglangjiang and Diandongnan buffalo, and 68 alleles unique to Binglangjiang buffalo). The genetic diversity parameters in Binglangjiang buffalo were much higher than that in the control groups, its mean number of alleles per locus, mean observed heterozygosity, mean expected heterozygosity and polymorphism information content(PIC) were 8.433 3, 0.640 5, 0.600 9 and 0.594 7, respectively. Inbreeding coefficient (FIS) in Binglangjiang buffalo was 0.061 9. Genetic distance(DA) between Binglangjiang and Murrah buffalo was the lowest(0.114 4), while genetic distance(DA) between Diandongnan and Binglangjian buffalo(0.382 9) or between Diandongnan and Murrah buffalo(0.555 3) was much larger than that between the two formers, which make Binglangjiang buffalo and Murrah buffalo, grouped together and Diandongnan independently clustered in the NJ tree. Structure analysis revealed that Binglangjiang buffalo displayed a genetic mixed pattern by river buffalo and swamp buffalo, most of the individuals having some proportion of swamp buffalo component (membership coefficients =0.067 2). The results indicate that Binglangjiang buffalo is of high degree of genetic diversity and is unique genetic resource, but this buffalo is under the conditions of heterozygote deficit and genetic mixture with swamp buffalo.