Abstract: Oncorhynchus mykis is an important reared freshwater species, which has been cultured for more than thirty years in Gansu Province, China. Due to the ecological conditions, different feeding manners, the various breeding goals and some native breeds had been formed in the past years, such as Gansu golden trout, which had past the National Aquaculture Species Identification Committee identifying and was recommend as the species reared in the northwestern areas. However, the genetic differentiation and the genetic variability are still unknown. In order to get insight into the genetic variability in its population and provide the genetic background and guidance for its conservation and utilization, 20 microsatellite DNA markers were applied to analyze genetic diversity and genetic differentiation.Genome DNA was extracted from each 50 individuals in Donaldson rainbow trout, Albino rainbow trout and Gansu golden trout in total 150 individuals. Each fish was extracted from ethanol-preserved caudal fin by using phenol-chloroform method. The PCR was performed in a 25 μL reaction and the products were isolated by electrophoresis on 10% polyacrylamide gel and visualized by silver staining. The data were calculated and analyzed by statistic method. 18 microsatellite primers were successfully amplified, 13 loci with reasonable polymorphism in the three populations. And there were a large range of genetic variation existing in Gansu golden trout. The number of alleles in these loci ranged from 2 to 7. 51 alleles was obtained; the mean number of alleles was 3.00; the value of mean polymorphism information content (PIC) was 0.440, the PIC value was more than 0.5 in locus AF346692, AF375011, AF352739, AF352746, AF352747, AF352751, AF352756, AF352763 and AF352765; the PIC value was more than 0.25 and less than 0.5 in locus AF352767 and AF352762; the PIC value was less than 0.25 in locus AF352750 and AF352749. The observed heterozygosity (Ho) and the expected heterozygosity (He) were calculated based on frequencies of genotypes and alleles of each microsatellite locus, the value of average observed heterozygosity (Ho) and expected heterozygosity (He) were 0.498,0.498 respectively, which showed that Gansu golden trout had relatively abundant genetic diversity.Genetic distance analysis showed that the largest distance was between Donaldson rainbow trout and Gansu golden trout and the smallest distance was between Albino rainbow trout and Gansu golden trout. Furthermore, it was found that some microsatellite loci of the three breeds deviated from Hardy-Weinberg equilibrium, where the majority of the variance in the Oncorhynchus mykiss was within populations (80.7%), and small proportion was among populations (19.3%). The medium level of overall genetic diversity and medium Fixation Index (Fst=0.193) indicated that the Oncorhynchus mykis reared in Gansu had relatively medium genetic variation. Obviously, the genetic characteristics and the genetic status of Gansu golden trout are getting stable. The genetic differentiation was significant among the three breeds, and selective breeding impacted genetic variations on the breeds. The results provided the guidance for its breeding, spreading, conservation and utilization.