The stable isotopes of nitrogen (d15N) and carbon (d13C) provide powerful tools for quantifying trophic relationships and carbon flow to consumers in food webs; however, the isotopic signatures of organisms vary within a lake. Assessment of carbon and nitrogen isotopic signatures in a suite of plants, invertebrates, and fishes in Lake Kyoga, indicated significant variation between two sites for d13C (paired t = 6.305; df = 14, P < 0.001 and d15N paired t = 1.292; df = 14; P < 0.05). The fish fauna in Bukungu was generally more 13C enriched (mean d13C = –16.37 ± 1.64&) than in Iyingo (mean d13C = –20.80 ± 2.41&) but more d15N depleted (mean d15N = 5.57 ± 0.71&) than in Iyingo (mean d15N = 6.92 ± 0.83&). The simultaneous shifts in phytoplankton and consumer signatures confirmed phytoplankton as the major source of carbon for the food chain leading to fish. Limited sampling coverage within lakes may affect lake wide stable isotope signatures, and the same error is transferred into trophic position estimation. Consideration of potential intra-lake spatial variability in isotope ratios and size is essential in evaluating the spatial and trophic structure of fish assemblages