Sixteen microsatellite markers were used to analyze the genetic diversity and population structure of Rutilus rutilus from the Beiwan section of the main stream, Habahe River and Ulungur Lake in the Irtysh River basin. The expected heterozygosity, average number of alleles and polymorphism information content were 0.916 8, 18.937 5 and 0.902 9, respectively, suggesting higher level of genetic diversity. The genetic diversity of fish in Ulungur Lake was relatively lower than that in Irtysh River. The principal component analysis showed that salinity might be the major factor for genetic diversity between Ulungur Lake and the other two sampling sites. Fst and UPGMA cluster showed that the three populations were almost non-differentiated. AMOVA analysis revealed a high genetic variation within individuals (88.47%), while the genetic variation among populations was low (1.83%), indicating lower variation levels among the three populations.