Stable isotope analysis has been extensively used as an effective tool in determination of trophic relationship in ecosystems. In freshwater ecosystem, aquatic invertebrates represent main component of a river food web. This study was carried out to determine potential food sources of freshwater organism together with pattern of trophic position along the river food web. In this study, rivers of Belum-Temengor Forest Complex (BTFC) has been selected as sampling site as it is a pristine area that contains high diversity and abundance of organisms and can be a benchmark for other rivers in Malaysia. Stable isotope ratios of carbon ( C) and nitrogen ( N) were applied to estimate trophic position and food web paradigm. Analysis of stable isotopes based on organic material collected from the study area revealed that the highest C value was reported from filamentous algae (− 22.68 ± 0.126 / ) and the lowest C was in allocthonous leaf packs (− 31.58 ± 0.187 / ). Meanwhile the highest N value was in fish (8.45 ± 0.177 / ) and the lowest value of N was in autochthonous aquatic macrophyte (2.00 ± 1.234 / ). Based on the N results, there are three trophic levels in the study river and it is suggested that the trophic chain begins with organic matter followed by group of insects and ends with fish (organic matter < insects < fish).