By the method of field investigation in combining with cluster analysis, multi-dimensional scaling analysis and canonical correspondence analysis, and using the indices of Shannon, Pielou, and Margalef, this paper studied the community structure and biodiversity of macrobenthos in the Chaohu basin in spring, and their relationships with environmental factors. A total of 23 macrobenthos species were collected, among which, six species (26.1% of total) were annelids, eight species (34.8%) were arthropods, and nine species (39.1%) were mollusks. The dominant species were Limnodrilus hoffmeisteri, Branchiura sowerbyi, and Bellamya aeruginosa, and their distribution had obvious spatial heterogeneity. The upper reaches of the rivers inside and out of Chaohu Lake had the highest macrobenthos biodiversity, followed by the Chaohu Lake and its estuary, and the lower reaches of the rivers inside and out of Chaohu Lake. The spatial distribution of the macrobenthos was also affected by various environmental factors, among which, Chl a and NO3(-) were the main ones.