Broadscale Distribution and Pronounced Mitochondrial Phylogeographic Structure in the Freshwater Minnow Opsariichthys bidens Across China's Major River Basins

China's complex river basins constitute a critical biogeographic setting for investigating genetic differentiation and lineage divergence in freshwater fishes. Opsariichthys bidens is broadly distributed among multiple, hydrologically isolated river basins, rendering it a well‐suited system for assessing mitochondrial phylogeographic structure across major river basins. Here, we used newly generated mitochondrial data to characterize broad‐scale genetic variation in O. bidens across China's major river basins. We analyzed 379 cytochrome b ( cyt b ) sequences sampled from 13 geographically distinct populations, including seven populations from the Yangtze River and one population from each of six other major rivers, complemented by complete mitochondrial genomes from one individual from each population. Mitochondrial diversity was high at the species level (haplotype diversity, H d = 0.942; nucleotide diversity, π = 0.02526), but varied markedly among populations, with some populations showing haplotype fixation and others retaining divergent haplotypes. Genetic differentiation was pronounced, with 83.84% of variation attributable to differences among populations; 93.6% of pairwise F st values exceeded 0.25. Haplotype‐network and phylogenetic analyses showed two deeply divergent mitochondrial lineages, broadly distributed in northern and southern China, respectively, with further substructure broadly associated with major river basins. Complete mitochondrial genomes exhibited high structural conservation across lineages, and the clearest lineage‐associated annotation differences were observed in the termination codons of nd1 and cox3 . Overall, these results provide a broad‐scale mitochondrial phylogeographic assessment of O. bidens and reveal a general association between river‐basin geography and mitochondrial lineage structure across China.