Phylogeographic Pattern and Genetic Structure of the Cyprinid Fish Microphysogobio kachekensis (Oshima 1926) in Mainland China and Hainan Island Based on Mitochondrial and Nuclear DNA

South China’s freshwater biodiversity has been shaped by Quaternary climatic oscillations and persistent geological barriers. We investigated the phylogeography and conservation implications of the primary freshwater fish Microphysogobio kachekensis across mainland China and Hainan Island using mitochondrial (cyt b and control region) and nuclear (RAG2 and rpS7-1) markers from 200 individuals. Mitochondrial analyses recovered two major lineages and multiple sublineages largely structured by drainage basins, whereas nuclear data resolved four geographically concordant lineages. Population differentiation was strong (high FST), and SAMOVA/AMOVA supported major barriers restricting gene flow, including the Qiongzhou Strait, Gulf of Tonkin, Yunkai Mountains, and Nanling Mountains. Ancestral-area reconstruction inferred the Pearl River region as the most likely source area, followed by dispersal to northern Hainan and subsequent expansion to southern Hainan and the Red River, with additional northward expansion to the Zhejiang–Fujian region. Despite high haplotype diversity, within-population nucleotide diversity was low, consistent with long-term river isolation and complex demographic history. We propose six ESUs and four MUs for evolutionarily informed conservation and to guide stock enhancement in southern China.