From (AAAG)(n) enriched genomic library, we developed 25 microsatellite loci in Rhinogobio ventralis, an endemic fish in the upper reaches of the Yangtze River in China, using the fast isolation by AFLP of sequences containing repeats (FIASCO) protocol. Twelve primer pairs exhibited polymorphic with two to 11 alleles per locus, while the rest 13 displayed monomorphic in a test on a population of 40 individuals. Observed heterozygosities ranged from 0.15 to 0.88. Nine loci are highly informative (PIC > 0.5). These loci should be useful in further genetic analyses in different populations of Rhinogobio ventralis and its congeneric species.