Animals that co-occur in a region (sympatry) may share the same environment (syntopy), and niche differentiation is expected among closely related species competing for resources. The masked booby (Sula dactylatra) and smaller congeneric red-footed booby (Sula sula) share breeding grounds. In addition to the inter-specific size difference, females of both species are also larger than the respective males (reversed sexual size dimorphism). Although both boobies consume similar prey, sometimes in mixed-species flocks, each species and sex may specialize in terms of their diet or foraging habitats. We examined inter- and intra-specific differences in isotopic values (δ13C and δ15N) in these pelagically feeding booby species during the incubation period at Clarion Island, Mexico, to quantify the degrees of inter- and intra-specific niche partitioning throughout the annual cycle. During incubation, both species preyed mainly on flyingfish and squid, but masked boobies had heavier food loads than red-footed boobies. There was no overlap in isotopic niches between masked and red-footed boobies during breeding (determined from whole blood), but there was slight overlap during the non-breeding period (determined from body feathers). Female masked boobies had a higher trophic position than conspecific males during breeding; however, no such pattern was detected in red-footed boobies. These results provide evidence of inter- and intra-specific niche partitioning in these tropical seabird species, particularly during the breeding period and in the more-dimorphic species. Our results suggest that these closely related species use different strategies to cope with the same tropical marine environment.