In the middle and lower reaches of the Yangtze River, there are 108 freshwater lakes with surface area larger than 10 km², accounting for 51.3% of the total freshwater lake area in China. However, few studies of macrozoobenthos were conducted at the regional scale, contrasting with many studies focused on individual or several lakes. To elucidate macrozoobenthic community structure and their relationships with spatial and environmental factors in shallow lakes along the Yangtze River, physicochemical parameters and macrozoobenthos were measured in 69 lakes of this region during summer in 2008 and 2009.We also evaluated the relative influences of local environmental conditions versus regional processes on macrozoobenthic community structure. The results showed that total dissolved solids (TDS), conductivity, nitrogen and phosphorus differed significantly among the five lake districts, but not for chemical oxygen demand (COD_Mn), chlorophyll a and Trophic State Index (TSI) index. In terms of density, 46 lakes (66.7% of the total 69 lakes) were dominated by Oligochaeta and Chironomidae, and Gastropoda was one of the dominant taxa in 16 lakes. With respect to biomass, 27 lakes were also dominated by Oligochaeta and Chironomidae, and 33 and 9 lakes were dominated by Gastropod and Bivlavia due to their relatively large body size, respectively. Canonical correspondence analyses (CCA) showed that macrozoobenthic community structure reflected the influence of local conditions (TSI, Macrophytes, Chl a, TDS) as well as regional processes. Only 33.9% of variation in a density matrix was explained by local environmental conditions and regional processes. Of the total explained variation, 48.1% and 35.4% were purely related to local environmental parameters and spatial variables, respectively. The high proportion of variation related to spatial variables indicated the importance of regional processes on spatial patterns of macrozoobenthic assemblages in this region.