<p>The ichthyofauna of the Perlis River basin, a critical wetland-associated region in Malaysia, was last comprehensively studied over a decade ago. Fish assemblages in the lower reaches, floodplains, and associated wetland habitats remain insufficiently documented. This study sought to compare fish assemblages across three distinct habitats within the wetland integral to the basin’s ecosystem and expand checklist of freshwater fishes in Perlis River basin. A total of 58 species from 25 families were recorded during surveys conducted between January and September 2022 for Perlis River basin. Cyprinidae was the most abundant family (13 species), followed by Bagridae, Osphronemidae, and Channidae, with four species each. Species richness was highest for the upstream 1, followed by downstream, Timah-Tasoh Reservoir (TTR), and upstream 2. Fish assemblages varied significantly between habitats, forming distinct groups based on habitat types, as indicated by 2D-nMDS. The taxonomic compositions for 20 sites were relatively similar, except for four sites, which fell outside the funnel plot. The formation of the reservoir reduced native fish species richness and led to similar taxonomic composition, suggesting potential biotic homogenization within the fish community. Upcoming investigations are critical to better understand the role of wetland ecosystems and floodplains in conserving native fish diversity, sustainable fisheries purposes and mitigating the impacts of invasive species.</p>