The origin and evolution of polyploid organisms have been extensively studied in plants, but this topic remains only partially understood in vertebrates, where polyploidy is relatively rare. In this study, we used Misgurnus anguillicaudatus, a fish that comprises five ploidy levels in nature, as a model animal to improve our understanding of biogeographic history and evolution of polyploid vertebrates. After collecting samples from different geographical populations in China, their ploidy levels were determined using flow cytometry. Two mitochondrial markers (cytb and CR) were then used for phylogeographic analyses to unravel the possible origins of diploids and tetraploids in China. The results showed that different ploidy specimens were not evenly distributed: diploids had wider geographic distribution than tetraploids and triploids. There was no clear allopatric geographical range or boundary to divide diploid and polyploid populations. Rather, the analysis of mtDNA sequences indicated that tetraploids were autopolyploids, with lower genetic diversity than diploids. The results suggest that tetraploids originated from sympatric diploids via multiple polyploidization events. The genetic structure showed similar patterns between diploids and tetraploids, whereas complex genetic differentiation was found among regions. The potential origin of M. anguillicaudatus was deduced to be in the Pearl River basin, which exhibited the highest nucleotide diversity and genetic differentiation. These findings provide insights into the evolution of polyploidy in vertebrates.