The Bighead Catfish (Clarias macrocephalus) plays crucial roles in the ecosystem and as a protein source for humans in Nong Han Lake, Thailand, but their ecological and population dynamics, along with their genetic diversity and structure, are poorly understood. Therefore, the dynamics of genetic diversity and differentiation of bighead catfish populations in Nong Han Lake were investigated during 2018–2023 to provide essential insights into preserving biodiversity and maintaining ecological balance. Microsatellite genotyping was used to assess the genetic diversity of one bighead catfish population in 2018, two populations in 2023, and one hatchery population. Potentially stable, effective population sizes and distinct genetic clusters of these populations were identified, which represented the resilience of populations and the complexity of their genetic dynamics. Genetic differentiation between sample dates and among populations suggested that limited recent and historical genetic exchanges could potentially influence genetic structure. The impact of ecological factors, including flooding, metapopulation dynamics, and human interventions, was indicated by unique and shared gene pools among populations and subpopulations in different years. Ongoing assessment of genetic diversity and structure of bighead populations is essential for developing conservation strategies and ensuring sustainable management.