China's five largest freshwater lakes (Poyang, Dongting, Taihu, Hongze, and Chaohu) are all located in lowland regions and differ greatly in lake morphology, hydrology, and water chemistry. However, basic knowledge of diversity patterns and factors driving macroinvertebrate community structure is lacking. We analyzed the composition and diversity of benthic macroinvertebrates and evaluated the roles of environmental factors and dispersal processes in determining beta diversity at both the lake and regional scales. The two Yangtze-connected lakes (i.e., Lakes Poyang and Dongting) were highest in taxonomic diversity. Differences in taxonomic composition were evident among the five lakes, with the two Yangtze-connected lakes dominated by bivalves and/or gastropods, while Lake Taihu showed great changes in taxonomic composition among sites in accordance with its high habitat heterogeneity. Lake Hongze was dominated by polychaetes, crustaceans, and bivalves. The eutrophic-hypertrophic Lake Chaohu was exclusively dominated by chironomids and oligochaetes. At the lake scale, total gamma-diversity was mainly attributed to beta (1)-diversity (among sites) ranging from 57.9 to 87.6%. The total gamma-diversity at the regional scale was primarily related to higher beta-diversity, with beta (1) and beta (2) (among lakes) accounting for 30.4 and 58.8% of total taxa richness, respectively. Environmental conditions appear to be important in structuring benthic macroinvertebrate communities at both the lake and regional scale. Dispersal does not appear to affect distribution of macroinvertebrates at the lake scale.