Background: Differences in the trophic niche often underlie ecological specialization of individuals and can promote ecological speciation of populations, but studies showing a link between differences in the trophic niche and genetic differentiation of populations are rare. On the island of San Cristóbal (Galapágos archipelago), a strong genetic differentiation between two relatively proximate populations (subspecies; Amblyrhynchus cristatus mertensi and A. c. godzilla) of marine iguanas along the coastline has been observed. Here, we explore whether this genetic differentiation is mirrored in the iguanas’ trophic niche. Results: Although, no significant difference in the number of consumed algal taxa between subspecies were detected, the Schoener index exhibited low diet overlap between A. c. mertensi and A. c. godzilla. The latter was also demonstrated by the PERMANOVA analysis with significantly different diet OTU composition from the fecal samples between subspecies. Stable isotope analysis revealed that subspecies identity was overall more important than site for the iguanas’ resource use. Conclusions: By applying a metabarcoding approach on feces samples in combination with stable isotope analysis of skin sheds, we found that A. c. mertensi and A. c. godzilla differ in their ecological niches. Moreover, stable isotope analysis indicated that marine iguana populations have low spatial foraging distances, which, together with the diet partitioning patterns, might explain, at least partially, the lack of gene flow between these geographically proximate marine iguana populations. Key words: diet analysis, trophic niche, metabarcoding, stable isotopes, marine iguanas, Amblyrhynchus cristatus