Benthic macroinvertebrates are vital in the food chain of aquatic bio-communities. Substrata play an important role in the benthic invertebrate communities, and the characteristics of stream substrata, such as bed material size, heterogeneity and stability, can obviously affect the benthic bio-community. Field investigations were done to study the biodiversity of macroinvertebrates in various stream substrata. Sampling sites differing in bed composition, latitude and climate were selected along the Yangtze River, the Yellow River, the East River, and the Juma River in China. The results show that the benthic community structures found in different substrata clearly differ, while those found in substrata of similar composition and flow conditions but in different macroclimates are quite similar. The study thus demonstrates that the benthic macroinvertebrate community is mainly affected by substrate composition and flow condition, but is generally unaffected by latitudinal position and macroclimate. Biodiversity in different substrata is assessed via several biodiversity indices. Taxa richness of the macroinvertebrate community was found to be highest in hydrophyte-covered cobbles, high in moss-covered bedrock, and relatively low in clay and cobble substrata devoid of plant biomass. Sand substrate is compact and unstable, and no benthic macroinvertebrates were found colonizing this substrate. Aquatic insects account for most of the macroinvertebrates found in these rivers. Different insect representatives dominate in different substrata types: mainly Ephemeroptera larvae were found in cobble and moss-covered bedrock substrata while Chironomidae larvae were found in clay beds. The relation between the number of species in the samples and the sampling area is also analyzed. The results support the species-area power rule. Since the experimental results differed little between surveys using 1m2 and 2m2 sampling areas, 1m2 is suggested as a sufficient minimum sampling area.