Extensive surveys were conducted to explore the diversity of fishes, distribution patterns, abundance, threat, and habitat status in the upper, middle, and lower stretch of river Gomti, a tributary of river Ganga. Altogether 56 fish species belonging to 20 families and 42 genera were collected from various sampling sites. Of the 56 species, five belong to the 'endangered' (EN) category and 11 belong to the vulnerable (VU) category. Six major categories of habitat were identified and pattern of fish assemblage and dominant genera in each habitat studied. Considerable differences were observed in the fish species richness and relative abundance (RA) of the species in the different sampling sites of river Gomti. Shannon-Wiener biodiversity index has been calculated for the fishes indicating considerable variation (p \ 0.05) across the river. Apart from Indian Major Carps (Labeo rohita, Catla catla, Cirrhinus mrigala), Chitala chitala, Notopterus notopterus, Ompok pabda, O. bimaculatus, Labeo bata, L. calbasu, Cirrhinus reba, Channa marulius, Bagarius bagarius, and Clupisoma garua were the important species. All the species have been reported for the first time in this river. Indiscriminate catch, poisoning, using of fine mesh sized nets, dumping of sewage, siltation, water abstraction, changing land use pattern, decreased water discharge, and exotic species threaten the fish diversity. Urgent need exists for taking up research on the priority fish species and their habitat. Restoration measures have been proposed based on ecosystem scale approach for fish biodiversity conservation.