To study the effect of Miaoling Mountain as a geographical barrier on the genetic structure and diversity of Siniperca scherzeri, the genetic diversity of seven wild S. scherzeri populations sampled from the Liujiang, Yuanjiang, and Wujiang River systems on both sides of Miaoling Mountain was examined by sequencing the mitochondrial DNA D-loop and analyzing their genetic variation and population dynamics. The results showed that out of 365 sequences of S. scherzeri, 57 polymorphic loci and 56 haplotypes were identified. The high-frequency haplotype hap3 was an ancestral haplotype shared by the three river systems. S. scherzeri in the Miaoling Mountain area can be divided into the northern (Yuanjiang and Wujiang Rivers) and the southern (Liujiang River) geographical populations. The southern region exhibited low genetic variability and a lack of genetic diversity, while the northern region displayed a high degree of variation and abundant genetic diversity. Significant genetic differentiation and barriers to genetic communication existed between the two geographical populations. Populations of S. scherzeri in Miaoling Mountain experienced demographic expansion in the early and middle of the MedioPleistocene period. The findings of this study indicated that the southern region had a small, closed population, and the original population structure of the ancestors was maintained due to an inbreeding depression effect, whereas the northern region had a large population with an ideal habitat; therefore, it had great potential for genetic evolution. It is inferred that after the population expansion of Siniperca chuatsi in the Middle Pleistocene interglacial period, it developed and evolved along different directions due to the barrier of Miaoling Mountain and differences in habitat, thus shaping the formation of two geographic populations with obvious genetic differentiation between the northern and southern regions. The southern population faces a survival crisis, as it can be seen as an evolutionarily significant unit. It is suitable for establishing a germplasm resource reserve of S. scherzeri in the upper Yuanjiang River system. The effects of the geographical barrier on the genetic structure and diversity of wild S. scherzeri populations in the Miaoling Mountain area were revealed at the molecular level, enriching the genetic background data of S. scherzeri in China and providing a theoretical basis for germplasm conservation and exploitation of S. scherzeri.