Null model analyses have greatly improved our understanding of species co-occurrence. Null model analyses have shown, for example, that cold-blooded animals show less segregated distributions than warm-blooded animals. This topic has rarely been studied simultaneously across multiple metacommunities. We analysed data on 10 stream metacommunities (with 10 communities in each metacommunity) of a cold-blooded animal group, benthic macroinvertebrates, and examined co-occurrence within five ecological guilds. We found that the segregated species co-occurrence was not the rule in stream invertebrate guilds. This was evidenced by the finding that only 10% of the 50 guild matrices we analyzed showed significant segregation and no matrices showed significant aggregation in the within-stream analyses. However, in the across-streams analysis, all guilds showed significant segregation. We neither found differences in the degree of segregation among the guilds, the degree of species segregation did not increase with overall environmental heterogeneity, and there were no differences in the relationships between species segregation and overall environmental heterogeneity among the guilds. Expanding the spatial extent from single stream metacommunities (i.e. within each stream) to the whole study region (i.e. across the streams) increased significantly segregation in all guilds. Because environmental heterogeneity across streams was much higher than within single streams, overall environmental heterogeneity may nevertheless have effects on species segregation. It also seems that the effects of overall heterogeneity on species segregation were masked by mass effects in the within-stream analyses.