Background <p><i>Neotricula aperta</i>, a freshwater snail found in the Mekong River, serves as the intermediate host of the blood fluke <i>Schistosoma mekongi</i>, the causative agent of schistosomiasis mekongi in Cambodia and Laos. Understanding the genetic diversity, population structure of this snail in relation to its geographical distribution is crucial for a comprehensive understanding of disease transmission. In this study, we investigated the genetic diversity, and genetic structure of <i>N. aperta</i> in Champasak Province, Laos.</p> Methods <p>A total of 80 <i>N. aperta</i> snails were collected from 13 various localities across five villages in Khong and Mounlapamok districts in Champasak Province, Laos in May 2024. Species of snails were initially identified based on morphology and subsequently confirmed by DNA barcoding. Molecular analyses were conducted using specific primers to amplify two mitochondrial DNA genes, namely cytochrome <i>c</i> oxidase subunit 1 (<i>cox1</i>) and 16S ribosomal RNA (16S rRNA), in order to assess the genetic diversity of <i>N. aperta</i> populations.</p> Results <p>Based on <i>cox1</i> sequences, the overall haplotype diversity was 0.996, while the overall nucleotide diversity was 0.049. For the 16S rRNA marker, the overall haplotype diversity was 0.911, and the overall nucleotide diversity was 0.015. Analysis of molecular variance (AMOVA) confirmed significant genetic differentiation (<i>P</i>-value &lt; 0.05) among populations at different spatial scales, including villages, catchments, and countries. This genetic structure likely reflects limited gene flow among snail populations, potentially due to geographical barriers. Although local environmental factors may also contribute to differentiation, the current genetic data are insufficient to distinguish between geographic isolation and adaptive divergence. Further ecological and functional investigations will be needed to determine whether adaptive processes are influencing population structure.</p> Conclusions <p>The genetic divergence of <i>N. aperta</i> observed in this study indicates a high level of genetic differentiation both among and within populations. This pattern suggests that <i>N. aperta</i> is possibly undergoing localized adaptation or that barriers to gene flow, such as physical, ecological, or behavioral factors, are promoting the accumulation of genetic differences. Micro-scale genetic structuring within populations may be driven by microhabitats or small-scale ecological gradients, while limited dispersal, localized mating preferences, or other behavioral traits may contribute to differentiation among populations.</p>