Abstract: Sinogastromyzon sichangensis is a small-size fish endemic to the upper Yangtze River mainly distributed in main stream and tributaries. Twenty-nine polymorphic microsatellite loci of the S. sichangensis were isolated by next-generation sequencing techniques. Four wild populations in Chishui River were amplified by 20 loci with mean alleles 14.5. The mean observed (H_o) and expected (H_e) heterozygosity were 0.620 and 0.882, respectively and the mean polymorphic information content (PIC) was 0.859. The H_o ranged from 0.520 (Maotai Town) to 0.669 (Chishui Town) and PIC ranged from 0.841 (Maotai Town) to 0.868 (Xishui River). The F_st value and analysis of population structure revealed a genetic differentiation between populations from Xishui River and the main stream of Chishui River. AMOVA showed that the genetic variation was 3.33% among populations while it was 96.67% within populations. These novel loci could use to investigate the population genetics and biological resource conservation strategy in S. sichangensis.