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In Malaysia, our knowledge of freshwater ecosystem and its aquatic inhabitants, particularly freshwater fish, remains inadequate, even in protected regions. It is essential to understand the composition of freshwater fish, their distribution along river gradients, and their interactions between environmental variables to develop and strategize effective conservation and management plans. Consequently, an investigation into freshwater fish assemblages in three rivers draining off from the western part of Endau-Rompin Landscape was conducted in 12 established substations. Sampling sessions were conducted from September to December 2021 in the Labis district of Johor, using multiple sampling methods along 200 meters for each substation. The environmental variables were measured using water quality parameters. A total of 66 species were collected. The family Cyprinidae presented the highest species diversity (17 species), constituting 52% of total capture. Crossocheilus obscurus was the most dominant species, and the highest species richness was recorded in the Segamat River (45 species), followed by Juaseh (36 species) and Labis River (34 species). It was discerned that the composition of fish varied between the substations. The knowledge presented here is the first documentation on the freshwater fish from these rivers. It would serve as a baseline information for key authorities and stakeholders to conserve the biodiversity inhabiting freshwater ecosystems in Malaysia.