Abstract: The population genetic diversity and genetic structure of non-native fish species are crucial for understanding their baseline conditions and formulating effective management measures. To investigate the genetic diversity of non-native fish species in the Irtysh River Basin, samples of three sympatrically distributed non-native fish species from 13 sampling sites were collected in the Irtysh River and Ulungur River, including a total of 79 individuals of Pseudorasbora parva, 33 of Cyprinus carpio, and 16 of Carassius auratus. Using the mitochondrial cytochrome b gene (CYTB) as a molecular marker, the genetic diversity and genetic structure of these three species were analyzed. The results showed that Pseudorasbora parva and Carassius auratus exhibited relatively high levels of genetic diversity, whereas Cyprinus carpio displayed a pattern of high haplotype diversity (Hd) but low nucleotide diversity (π). Carassius auratus showed the highest haplotype diversity (Hd = 1.000 in the Irtysh River group) and relatively high nucleotide diversity (π = 0.012 87). Pseudorasbora parva maintained high genetic diversity in both river systems (Hd = 0.834–0.842, π = 0.010 90–0.011 48). Cyprinus carpio had relatively high haplotype diversity (Hd = 0.697–0.764) but generally low nucleotide diversity (π = 0.003 92–0.004 04). Inter-population comparisons revealed that genetic diversity of Pseudorasbora parva and Cyprinus carpio was higher in the Ulungur River than in the Irtysh River, whereas Carassius auratus showed the opposite trend, with significantly lower genetic diversity in the Ulungur River group than in the Irtysh River group. Haplotype network analysis indicated that both the Irtysh River and Ulungur River groups of Pseudorasbora parva, Cyprinus carpio, and Carassius auratus possessed unique haplotypes. Analysis of molecular variance (AMOVA) showed that most of the genetic variation in Pseudorasbora parva, Cyprinus carpio, and Carassius auratus occurred within populations. Neutrality tests (Fu’s Fs and Tajima’s D) indicated no significant population expansion in any of the studied groups of the three species. Finally, some recommendations were provided, including regulating fisheries activities and aquaculture transportation, strictly prohibiting the disorderly introduction of non-native groups across watersheds, and continuously conducting genetic monitoring to reduce the risks of genetic mixing and biological invasion.