Abstract Freshwater fish ecology has greatly benefited from the use of innovative tools such as stable isotope analysis to determine the ecological effects of non-native fishes. Stable isotope analyses are based on the predictable relationship between the isotope composition of a consumer and its prey, and have become increasingly popular amongst aquatic ecologists. In parallel, they have been implemented as a sensitive, cost-effective and temporally integrative tool to analyse the trophic interactions between native and non-native species, and to detect some subtle ecological effects of human activities, such as the introduction of non-native freshwater fish species. This review aimed to understand how stable isotope analyses have been used and how they have provided new insights into the ecological impacts of non-native freshwater fishes. Specifically, the published literature (45 articles) was reviewed to establish the current state-of-the-art. The use of stable isotope analyses in the field is still an emerging approach. The majority of studies were conducted on lentic ecosystems in North America targeting three main families of non-native fish species. Measurements were most commonly made with carbon and nitrogen stable isotopes using muscle samples. The most recent theoretical and methodological advances were illustrated by selecting some case studies conducted with different non-native species and biotic interactions. Finally, several recommendations for an optimised use of stable isotope analyses for freshwater ecological studies related to trophic interactions of non-native freshwater fish species were established.