Abstract: In October 2011, an investigation was conducted on the macrobenthos at 45 sampling sites in the Weihe River basin of Northwest China. A total of 116 macrobenthos species were collected, of which, 91 species (78.4%) were aquatic insects, 12 species (10.3%) were mollusks, 9 species (7.8%) were annelids, and 4 species (3.4%) were crustaceans. According to the species composition and relative abundance of macrobenthos, the 45 sampling sites could be divided into three groups by using twoway indicator species analysis (TWINSPAN) and detrended correspondence analysis (DCA). In the first group, Serratella sp., Hydropsyche sp., and Antocha sp. were the indicator species. In the second group, Tabanus sp., Alotanypus venustus, one species of Pelecorhynchidae, Liodessus sp., and Limnodrilus hoffmeistteri were the indicator species. In the third group, Ephemera nigroptera and Polypylis hemisphaerula were the indicator species. The canonical correspondence analysis (CCA) indicated that ‘cobble + boulder’ type substrate, current velocity, water conductivity, water depth, and water total nitrogen content had significant effects on the spatial distribution of macrobenthos in Weihe River basin.