Objective To investigate the genetic diversity, genetic differentiation and phylogenetic relationships of Anopheles sinensis populations in different ecological regions of Anhui Provinc. Methods Adult An. mosquitoes were captured from Huangshan District, Nanling County, Feidong County, Dingyuan County, Suixi County and Guzhen County that were randomly selected from three ecological regions of north of the Yangtze River, areas between the Yangtze River and Huaihe River and north of the Huaihe River from 2023 to 2024. Following morphological identification, genomic DNA was extracted from mosquitoes, and the ribosomal DNA internal transcribed spacer 2 (rDNA-ITS2) and mitochondrial cytochrome c oxidase subunitⅠ(COⅠ) genes were amplified using PCR assay. Following bidirectional sequencing of the amplified products, the yielded sequences were subjected to BLAST alignment in the National Center for Biotechnology Information (NCBI). The obtained sequences were aligned using the software MEGA 11. The inter-population genetic distance was calculated, and phylogenetic trees were built. The polymorphism information content of An. sinensis populations was calculated using the software DnaSP 5 and mismatch analysis was performed. Analysis of molecular variance (AMOVA) and neutrality tests were performed using the software Arlequin 3.5.2.2, and the genetic differentiation (F_ST) and gene flow (Nm) values were calculated. In addition, the SPSS 18.0.0 software was used to generate scatter plots and examine the correlation between inter-population geographical distance and genetic distance. Results A total of 300 An. sinensis samples were obtained. After PCR amplification of the rDNA-ITS2 sequence, a specific band appeared at approximately 490 bp in length, which was consistent with the size of An. sinensis (GenBank accession number: MG816544.1). Following PCR amplification of the COⅠsequence, BLAST alignment showed that the sequence had a 99% identity with that of An. sinensis. The A + T content (68.8%) was higher than the G + C content (31.1%) in the COⅠgene sequence, showing a remarkable AT bias. A total of 129 polymorphic sites and 204 haplotypes were identified, including 166 unique haplotypes and 38 shared haplotypes. The haplotypes Hap_11, Hap_12, Hap_15, Hap_26, and Hap_38 were detected in the COⅠgene sequences of mosquitoes from all three ecological regions, with Hap_26 shared by the most populations and present in all six counties (districts). The haplotype diversity was 0.994, and the nucleotide diversity was 0.010 1. Among the six counties (districts), Nanling County had the highest values in terms of numbers of polymorphic sites and haplotypes, haplotype diversity, and nucleotide diversity. The results of AMOVA showed that the intra-population variation of An. sinensis was 97.5% in six counties (districts), and the inter-population variation was 2.5% (P < 0.01). The F_ST values of An. sinensis were all < 0.1 in six counties (districts), and the Nm values were all > 1. The Tajima’s D values of An. sinensis populations were all negative in six counties (districts), with P values of < 0.05 for Tajima’s D values in Guzhen County, Huangshan District, and Nanling County, and the Fu’s Fu test also supported the results of Tajima’s D test. The mismatch distribution of An. sinensis populations all showed an obvious multimodal structure in six counties (districts). In addition, there was no correlation between the genetic distance and geographical distance of An. sinensis collected from six counties (districts) (r² = 0.109, P > 0.05). Phylogenetic analysis revealed no geographical clusters of An. sinensis collected from the six counties (districts). Conclusion There are high genetic polymorphisms, sufficient intra-population gene flow and no remarkable genetic differentiation in An. sinensis populations in different ecological regions of Anhui Province; however, population expansion has been identified recently.