Hybridization between native and non-native species has consequences for survival and growth rates of hybrid offspring, but the influences on their functional roles such as habitat use are little studied and poorly understood. The Japanese native common carp Cyprinus carpio coexist and hybridize with non-native domesticated carp in natural Japanese lakes. We have combined stable isotope and molecular information to examine whether habitat use of carp varies depending on the degree of hybridization between native and non-native carp. We sampled 69 carp from Lake Kasumigaura where hybrid swarms between native and non-native carp are advancing, evaluated the degree of hybridization for each individual by genotyping five single nucleotide polymorphism (SNP) markers, and analyzed their carbon and nitrogen stable isotopes. Although we did not find any genetically pure native carp in the lake, the results showed that carp δ13C increased with increasing frequency of non-native alleles but that δ15N did not change. This indicates that non-native carp use the littoral zone more frequently than native carp. This difference in habitat use was supported by a multisource mixing model, showing that the contribution of limnetic primary consumers to the diets of non-native carp was lower than that of individuals with the highest frequency of native alleles. By combining two very different methods, our results thus suggest that multiple-generation hybridization can influence habitat and resource use. Habitat partitioning should be considered when evaluating the genetic impacts of invasive species and races on native species and ecosystem processes.