Diet composition and its ecological drivers are rarely investigated in coexisting closely related species. We used a molecular approach to characterize the seasonal variation in diet composition in four spiny lizard species inhabiting a mountainous ecosystem. DNA metabarcoding revealed that the lizards Sceloporus aeneus, S. bicanthalis, S. grammicus, and S. spinosus mostly consumed arthropods of the orders Hemiptera, Araneae, Hymenoptera, and Coleoptera. The terrestrial lizards S. aeneus and S. bicanthalis mostly predated ants and spiders, whereas the arboreal–saxicolous S. grammicus and saxicolous S. spinosus largely consumed grasshoppers and leafhoppers. The taxonomic and phylogenetic diversity of the prey was higher during the dry season than the rainy season, likely because reduced prey availability in the dry season forced lizards to diversify their diets to meet their nutritional demands. Dietary and phylogenetic composition varied seasonally depending on the species, but only dietary composition varied with altitude. Seasonal dietary turnover was greater in S. spinosus than in S. bicanthalis, suggesting site-specific seasonal variability in prey availability; no other differences among species were observed. S. bicanthalis, which lives at the highest altitude in our study site, displayed interseasonal variation in diet breadth. Dietary differences were correlated with the species’ feeding strategies and elevational distribution, which likely contributed to the coexistence of these lizard species in the studied geographic area and beyond.