Endemic fish species have long supported the livelihoods of local communities in the Rufiji River Basin (RRB). However, destructive fishing practices have led to a concerning decline in endemic fish stocks. To assess these changes, this study employed key informant interviews, focus group discussions (FGDs), and fishery surveys to assess the historical and contemporary distribution of endemic fishes within the RRB. DNA barcoding was also used to verify species identities. Out of 37 reported fish species, 33 species (54.55% endemic and 45.45% exotic to RRB) were confirmed through DNA barcoding and morphological characteristics. About 5 species including, Heterobranchus longifilis, Citharinus congicus, Labeo congoro, Mormyrus longirostris, and Labeobarbus leleupanus were rarely found in the field, despite being classified as Least Concern by IUCN. Additionally, five species that were reported to be present in the RRB by experienced fishers were not captured during sampling. This highlights the need for validation of the existence of such species through eDNA metabarcoding. Moreover, due to the rarity of some species in the area, their IUCN assessment should be revisited.