We investigated whether isotopic niche partitioning could mediate the coexistence between native (Hoplias sp. B) and non‐native (Hoplias mbigua) trahira in a Neotropical floodplain. We hypothesised that during the dry season both species inhabit isolated sites (i.e. lakes) and thus rely on distinct food resources to sustain their coexistence. We found evidence of trophic segregation among native and non‐native freshwater fish in the Upper Paraná River, Brazil. Even though both species rely on the same primary littoral carbon sources and present similar trophic diversity, trophic segregation was attained through larger niche amplitude and smaller isotopic niche width for the non‐native fish. Three decades after initial invasion, non‐native Hoplias have distinct foraging behaviour and do not compete for trophic resources with native trahira; this is likely due to morpho‐anatomical differences. Limited morphological similarity between these congeneric species might be the strongest factor contributing to their coexistence in the Paraná River floodplain during the dry season.