Animal interactions are an integral part of a community’s function, with influences ranging from the spatiotemporal habitat use of species to population effects to ecosystem management. Numerous non-native species are established or maintained through stocking in freshwater ecosystems with the potential to affect restoration of native species. Using acoustic telemetry, this study quantified the spatiotemporal co-occurrence of the native top-predator lake trout (Salvelinus namaycush) with non-native Chinook salmon (Oncorhynchus tshawytscha) in Lake Ontario over 2.5 years. Core home range overlap was observed during the summer, with depth acting as a mechanism of segregation, but with potential for interactions during vertical exploration. Fine-scale individual pairwise interactions confirmed the home range results. No horizontal overlap was observed during the winter and spring, but confidence was lower due to poor instrument coverage in deeper water, which the two species may frequent in these seasons. These results demonstrate the importance of depth in understanding fish interactions and highlight the usefulness of considering pairwise species interactions for understanding ecosystem community function to resource managers with multiple projects involving both native and non-native species.