Ecosystems are being disrupted by climate change and habitat fragmentation, which affect species survival through altered mating, feeding, and migration patterns. Mae Hong Son Province, Thailand, harbors a unique hydrological network that supports rich freshwater fish biodiversity. Blue mahseer (Neolissochilus stracheyi), which is restricted to headwater streams in Mae Hong Son, is particularly sensitive to habitat disturbances and has experienced population decline. Despite their vulnerability to climate change and habitat fragmentation, information on the genetic diversity, population structure, and environmental drivers of their distribution remains limited. In this study, microsatellite genotyping and mitochondrial DNA displacement loop sequence analysis were used to assess the genetic diversity and population structure of five blue mahseer populations in Mae Hong Son, with the aim of identifying reliable conservation units for effective management. Low genetic diversity levels across populations were identified (expected heterozygosity = 0.452 ± 0.037; allelic richness = 3.150 ± 0.506) with no evidence of inbreeding or outbreeding. A forecasted drop in heterozygosity below 0.1 within 50 years indicated the urgency of conservation attention. The five blue mahseer populations were clustered into three evolutionarily significant units (ESUs) based on historical isolation, phylogenetic distinctness, and significant genetic differentiation. Habitat suitability was assessed using MaxEnt species distribution modeling, which identified distance to rivers and annual mean total precipitation as significant environmental variables. The correlation between genetic differentiation and geographical distance suggested that habitat conditions primarily influence population genetic structure. Stocking between ESUs with differing genetic stocks is discouraged to avoid negative genetic effects. A comprehensive understanding of blue mahseer population dynamics, informed by the integration of genetic and ecological data, is needed to inform conservation strategies for resource management in Mae Hong Son.