Hokkaido Island is inhabited by two salamander species, Siberian salamander (Salamandrella keyserlingii) and Ezo salamander (Hynobius retardatus), which were previously considered incompatible to cohabit due to the predation and/or competition between them. Both species had never been seen reproducing sympatrically until a recent discovery in a pond where the two species spawn concurrently. We investigated their potential interspecific interaction, by identifying the trophic niche of the coexisting populations in this pond using isotope analysis. We initially conducted aquarium experiments to determine the trophic discrimination factor (TDF) of main-body δ values, which are required to accurately interpret the isotope ratios in the field. The TDF of δ¹⁵N and δ¹³C were 1.6‰ and 0.1‰ for S. keyserlingii and 1.5‰ and 0.5‰ for H. retardatus, respectively. The δ¹⁵N and δ¹³C results from the pond in June suggested the possibility that H. retardatus larva may prey on S. keyserlingii larva, but this prey–predator interaction had subsided by July, if ever. Further, the interspecific variations in δ values suggested that the two species consumed different foods in July, when both species reached isotopic steady states. Thus, we conclude that the coexisting salamander larvae do not have continuous prey–predator or competitive interaction as expected from their exclusive distribution patterns. The unsustainable negative interspecificity may be avoided due to microhabitat segregation in the pond or by a decrease in the S. keyserlingii population density after predation.