Abstract: Gobiocypris rarus is a small freshwater fish endemic to upper reaches of the Yangtze River. In China, the fish is being used as an aquatic laboratory animal mainly applied in researches on toxicology, fish pathology and genetics. Due to habitat loss and human activities, G. rarus is also considered as an endangered species listed in the China Red Data Book of Endangered Animals. Closed colony is an animal stock propagated by non-inbred within the stock, in which no genes are introduced from outside the stock from generation to generation. Because of high heterozygosity and stable genetic structure, closed colony animals are widely used in various researches. Therefore, foundation of a closed colony of G. rarus is the basis for its future laboratory use as well as species conservation. In the present study, we established a closed colony of G. rarus with 50 pairs of wild fish (P0) gathered in Hanyuan County, Sichuan Province. The stock propagated by maximum avoidance of inbreeding system for 4 generations (F1 to F4) was named as Ihb: IHB. Eleven microsatellite loci were used to monitoring the genetic variation among the generations. A total of 57 alleles were detected in the overall sample, while the number of alleles per locus, the effective number of alleles and the polymorphic information (PIC) averaged 5.2, 3.3 and 0.5282, respectively. For different generations of the closed colony, average expected heterozygosity (He) ranged from 0.5553 to 0.5742, and average PIC ranged from 0.5060 to 0.5318. No significant differences (P>0.05) on genetic diversity indices were detected among generations, including the averaged observed number of alleles (Na), average effective number of alleles (Ne), average observed heterozygosity (Ho), average expected heterozygosity (He) and average polymorphic information (PIC). Pairwise genetic similarities were over 0.99, implying closer genetic relationship and high genetic identity among generations. Analysis of molecular variance (AMOVA) revealed pairwise fixation index (FST) ranged from 0.0001 to 0.0031 with the overall FST value 0.0008 among generations, indicating that most of the genetic variance was among individuals rather than that of generations. Fisher's exact test on allele frequency showed no significant difference on any loci in successive generations (P>0.05). As a result, the genetic variation during the propagation of generations of G. rarus was successfully avoided and then high genetic diversity of the wild population was maintained in the strain. In other words, it indicated that Ihb: IHB of G. rarus fit the genetic quality of closed colony animal. Continuous reproductive strategy and genetic monitoring program should be carried out in order to use this newly established closed colony.