[Objectives] Mastacembelus armatus is a significant economic fish species in South China. However, there are few studies on the genetic diversity and population structure of M. armatus in China. Studying the genetic diversity and historical population dynamics of M. armatus can provide robust insights and data support for the conservation of its germplasm resources. [Methods] To investigate the genetic diversity and historical population dynamics of M. armatus in six regions of Guangdong Province, this study revealed the D-loop region sequences of 193 ind (Chaozhou:32; Heyuan:33; Shaoguan:30; Fengkai:30; Yangchun:32; Gaozhou:36) by PCR amplification and sequencing. Then, the population genetic diversity of M. armatus was analyzed through approaches including genetic diversity analysis, genetic differentiation analysis, neutrality tests, and Bayesian skyline plots. [Results] Sequencing results indicated that the D-loop region sequences were 871 ~ 872 bp in length, with the A + T content of 68.1% (Table 2). Genetic diversity analysis revealed that the six populations defined a total of 27 haplotypes, exhibiting high overall genetic diversity (H_p = 0.829), while the genetic diversity of the Chaozhou (H_p = 0.181 ± 0.090), Heyuan (H_p = 0.430 ± 0.108), and Yangchun (H_p = 0.179 ± 0.088) populations was low (Table 3). The genetic differentiation analysis revealed that the differentiation index F_st for the majority of populations was greater than 0.25, and the gene flow N_m was below 1, suggesting significant genetic differentiation among populations (Table 5). The neutral test indicated that the test values for the Chaozhou and Heyuan populations were negative (P<0.05) (Table 8), and the nucleotide mismatch distribution exhibited a single peak, suggesting population expansion, whereas the other populations adhered to the neutral evolution model (Fig. 3). The extended Bayesian skyline plot for the six populations together indicated historical reduction of M. armatus population. However, the population is presently in a stage of expansion (Fig.4). [Conclusion] The findings indicate that the overall genetic diversity of M. armatus throughout the six regions in Guangdong is substantial and increasing. Nevertheless, several populations exhibit low genetic diversity, necessitating the implementation of specific conservation measures.