Both environmental heterogeneity and mode of dispersal may affect species co-occurrence in metacommunities. Aquatic invertebrates were sampled in 20–30 streams in each of three drainage basins, differing considerably in environmental heterogeneity. Each drainage basin was further divided into two equally sized sets of sites, again differing profoundly in environmental heterogeneity. Benthic invertebrate data were divided into three groups of taxa based on overland dispersal modes: passive dispersers with aquatic adults, passive dispersers with terrestrial winged adults, and active dispersers with terrestrial winged adults. The co-occurrence of taxa in each dispersal mode group, drainage basin, and heterogeneity site subset was measured using the C-score and its standardized effect size. The probability of finding high levels of species segregation tended to increase with environmental heterogeneity across the drainage basins. These patterns were, however, contingent on both dispersal mode and drainage basin. It thus appears that environmental heterogeneity and dispersal mode interact in affecting co-occurrence in metacommunities, with passive dispersers with aquatic adults showing random patterns irrespective of environmental heterogeneity, and active dispersers with terrestrial winged adults showing increasing segregation with increasing environmental heterogeneity.