Dams interrupt the longitudinal connectivity of rivers by impeding the movement of water, sediments and organisms, which, in turn, could affect aquatic biodiversity and food web ecology. Using stable isotope analysis, we examined spatiotemporal variation in food web structure at four sites in the upper Yeilrmak River, Anatolia Peninsula, Turkey, in relation to environmental parameters and a dam. It was apparent that the dam created discontinuity in the longitudinal fluvial gradient of fish species richness, with more species observed at upstream sites. Fish assemblages from different sites and seasons occupied distinct areas of isotopic space. Isotopic niche space, trophic diversity, variation in 13C of basal resources and assemblage redundancy were all higher for the fish assemblage at the site downstream from the dam compared with the site above the dam, a possible indication of greater interspecific dietary variation. Food chain length (the range in 15N) was lower at the downstream site, possibly resulting from a greater tendency towards omnivory. The findings strongly suggest that the dam affects not only environmental conditions and fish diversity, but also trophic ecology. The results of the present study emphasise the need for research to assess potential effects of new dams in Anatolia on aquatic communities and ecosystem dynamics in rivers.