Invasive species can cause disruption of the trophic niche of resident species such that patterns of energy flow through ecological systems are altered. Quagga mussel Dreissena rostriformis bugensis Andrusov, 1897, a notorious invasive species, was introduced to Lake Powell (Utah and Arizona, USA) in 2012 and colonized the entire reservoir by 2017. To help understand potential effects of this invasive species on the trophic niche of fishes in Lake Powell, we characterized trophic niche of fishes and other organisms, as well as sources of variation in trophic niche, using stable isotope analysis of carbon and nitrogen prior to full colonization by quagga mussels. Trophic niche positions of fishes and other organisms in Lake Powell were consistent with other large lake or reservoir systems, consisting of 4 trophic levels and pelagic and littoral energy pathways. Trophic niche size decreased with increased trophic level among fishes, and there was high overlap in trophic niche among top predators. Trophic niche of fishes varied between northern and southern regions of the lake, among seasons, and, in some species, with body size. Basal trophic species (algae and chironomids) varied little between northern and southern regions and across years. Quagga mussels occupied a position in the food web where they were likely to directly impact the pelagic energy pathway, but resulting effects on trophic niche of fishes may be variable between regions of the lake.