Niche theory suggests that the coexistence of ecologically similar species at the same site requires some form of resource partitioning that reduces or avoids interspecific competition. Here, we investigated the temporal and spatial niche differentiation of two sympatric congeneric spiders,Peucetia rubrolineataandP. flava, inhabitingTrichogoniopsis adenantha(Asteraceae) plants along an altitudinal gradient in various shaded and open areas in an Atlantic forest in Serra do Japi, SP, Brazil. In theory, the coexistence of twoPeucetiaspecies could be explained by: (1) temporal segregation; (2) differential use of the branches of the plant; (3) differential use of specific parts of the branches of the plant; (4) differential distribution in shaded and open areas; and (5) differential altitudinal distribution of the twoPeucetiaspecies. With respect to temporal niche, we observed that the two spider species had a similar age structure and similar fluctuation in abundance throughout the year. With respect to micro-habitat use, in both species, different instars used different plant parts, while the same instars of both species used the same type of substrate. However, the twoPeucetiaspecies segregated by meso-habitat type, withP. rubrolineatapreferringT. adenanthaplants in shaded areas andP. flavapreferring those in open areas. Our results support the hypothesis of niche partitioning begetting diversity, and highlight the importance of analysing habitat use at multiple scales to understand mechanisms related to coexistence.