<p>The genetic diversity and differentiation of Chinese sleeper <i>Perccottus glenii</i> populations are estimated based on ISSR markers for the populations inhabiting the water bodies of the Volga and Irtysh river basins. In the studied part of the Volga River basin, the proportion of polymorphic loci (<i>P</i>) of Chinese sleeper was 80.4%, and the genetic diversity (<i>h</i>) was 0.26. All studied Chinese sleeper populations in the Kuibyshev Reservoir watershed area (Ulyanovsk oblast, Samara oblast, city of Tolyatti; Russia) are weakly differentiated genetically, originating from a single source of invasion. The indicators of polymorphism and genetic diversity of Chinese sleeper in the water bodies of the Irtysh River basin (<i>P</i> = 98.2%, <i>h</i> = 0.33) are higher than that in the Volga River basin. Genetic differentiation is weakly expressed (<i>G</i>_ST = 0.07) between the populations of Ipkul’ Lake and the Tobol River (Tyumen oblast, Russia); gene flow between these areas is significant, which indicates their recent divergence or the origin of one population from another. Genetic differentiation is high (<i>G</i>_ST = 0.31) between the populations of Berezovo Lake (Kurgan oblast, Russia) and Maloe Ploskoe Lake (Kazakhstan); this may indicate different sources of Chinese sleeper invasion in southern Siberia and northern Kazakhstan. Data on higher polymorphism of Chinese sleeper in the Tobol River (Irtysh River basin) and significant genetic differentiation between the Chinese sleeper populations of the Volga and Irtysh river basins (<i>G</i>_ST = 0.40) confirm only partially the generally accepted hypothesis about the colonization of Siberia by the Chinese sleeper through the South Ural invasion pathway, indicating the presence of several heterogeneous sources of Chinese sleeper invasion in the Ob’–Irtysh River basin.</p>