Spider species respond differently to variations in habitat structure; thus, differences in habitat structure may be responsible for variations in species composition of assemblages. However, little information exists on patterns of variation in spider species composition in tropical rainforests. We collected spiders and measured five different microhabitat characteristics in 20 sampling plots distributed among secondary and primary forest patches in an Atlantic rainforest, Brazil. Using multivariate analysis (non-metric multidimensional scaling - NMS), we checked for the existence of non-random patterns in the species composition of aerial (AG) and ground (GG) macroguilds, respectively. We also explored the relationships of those patterns with gradients in microhabitat characteristics and the influence of forest type (primary or secondary forest). We detected non-random patterns in spider species composition unrelated to microhabitat characteristics but differing between primary and secondary forest plots for both macroguilds. We discuss possible implications for studies of spider species composition and spider conservation in tropical forests.