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Background: The use and partitioning of trophic resources is a central aspect of community function. On the ground of tropical forests, dozens of ant species may be found together and ecological mechanisms should act to allow such coexistence. One hypothesis states that niche specialization is higher in the tropics, compared to temperate regions. However, trophic niches of most species are virtually unknown. Several techniques might be combined to study trophic niche, such as field observations, fatty acid analysis (FAA) and stable isotope analysis (SIA). In this work, we combine these three techniques to unveil partitioning of trophic resources in a tropical and a temperate community. We describe patterns of resource use, compare them between communities, and test correlation and complementarity of methods to unveil both community patterns and species’ niches. Methods: Resource use was assessed with seven kinds of bait representing natural resources available to ants. Neutral lipid fatty acid (NLFA) profiles, and d15N and d13C isotope signatures of the species were also obtained. Community patterns and comparisons were analyzed with clustering, correlations, multivariate analyses and interaction networks. Results: Resource use structure was similar in both communities. Niche breadths (H′) and network metrics (Q and H2′) indicated similar levels of generalization between communities. A few species presented more specialized niches, such as Wasmannia auropunctata and Lasius fuliginosus. Stable isotope signatures and NLFA profiles also indicated high generalization, although the latter differed between communities, with temperate species having higher amounts of fat and proportions of C18:1n9. Bait use and NLFA profile similarities were correlated, as well as species’ specialization indices (d′) for the two methods. Similarities in d15N and bait use, and in d13C and NLFA profiles, were also correlated. Discussion: Our results agree with the recent view that specialization levels do not change with latitude or species richness. Partition of trophic resources alone does not explain species coexistence in these communities, and might act together with behavioral and environmental mechanisms. Temperate species presented NLFA patterns distinct from tropical ones, which may be related to environmental factors. All methods corresponded in their characterization of species’ niches to some extent, and were robust enough to detect differences even in highly generalized communities. However, their combination provides a more comprehensive picture of resource use, and it is particularly important to understand individual niches of species. FAA was applied here for the first time in ant ecology, and proved to be a valuable tool due to its combination of specificity and temporal representativeness. We propose that a framework combining field observations with chemical analysis is valuable to understand resource use in ant communities.