[Objectives] Gobiobotia meridionalis is a small-sized bottom-dwelling endemic fish in China. There is still relatively few relevant genetic information for G. meridionalis. This study aimed to perform analysis of the genetic diversity and population structure of G. meridionalis from the Ganjiang River and Fuhe River. [Methods] In this study, we sequenced the cytochrome b (Cyt b) gene for 110 individuals of G. meridionalis from three geographical populations in the Ganjiang River and the Fuhe River. Population genetic diversity was measured for all samples and sampling groups in the DnaSP 5.0 software. Phylogenetic analysis of all haplotypes was conducted using the neighbor-joining (NJ) and the maximum likelihood (ML) based on the Kimura 2-parameter distance method in MAGA 7.0. A haplotype network was constructed based on median-joining method in PopART. Pairwise Fst values were calculated and analysis of molecular variance (AMOVA) was conducted by Arlequin v3.1. Statistical tests for neutrality analysis, including Tajima’s D and Fu’s Fs, and nucleotide mismatch distribution were conducted by Arlequin v3.1. [Results] A total of 45 haplotypes were identified for 110 individuals of G. meridionalis. The haplotype diversity (Hd) and nucleotide diversity (Pi) were 0.967 ± 0.006 and 0.007 1 ± 0.000 2, respectively. Haplotype diversity and nucleotide diversity of G. meridionalis were 0.911 ± 0.022 and 0.004 3 ± 0.000 2 in Fuzhou population, 0.950 ± 0.015 and 0.003 7 ± 0.000 2 in Baqiu population, 0.810 ± 0.130 and 0.002 6 ± 0.000 5 in Wanan population, respectively (Table 1). Phylogenetic and network topology showed that there was no distinct geographical distribution pattern (Fig. 2, Fig. 3). Genetic differentiation index (Fst) indicated high genetic differentiation between Fuzhou and Baqiu populations. Analysis of molecular variance (AMOVA) indicated that genetic variations between different geographical populations in the Ganjiang River and the Fuhe River were mainly within group (Table 2). The neutrality test indicated that the population expansion had occurred in Baqiu population about 0.38 million years ago, and Fuzhou population about 0.63 million years ago (Table 3). [Conclusion] The total genetic diversity of G. meridionalis is relatively high, but genetic differentiation exists in the Gangjiang and Fuhe population. Therefore, we suggest that the protection of G. meridionalis should be divided in the local population.