Knowledge of macroinvertebrate distributions and associated environmental drivers in subtropical Asian rivers is relatively scarce. To fill this knowledge gap, we examined the spatial variation of macroinvertebrate community structure and associated environmental conditions in a subtropical river system, the Dongjiang River Basin, in southeastern China. A total of 70 families and 9 classes of macroinvertebrates were identified from 74 sites sampled in January 2013. Our study has the following findings: (1) a distinct spatial differentiation of macroinvertebrate communities was present in the Dongjiang River Basin indicated by non-metric multidimensional scaling (NMDS), which corresponded to the northern region (NR), middle region (MR), and southern region (SR) gradient; (2) ANOVAs showed that diversity indices (total taxa, Margalef index and the Shannon diversity index), biotic indices (richness of EPT, percentage of EPT, and family biotic index) and most of the studied environmental conditions (elevation, slope, steam order, water temperature, electrical conductivity, dissolved oxygen, pH, substrates, ammoniacal nitrogen, total phosphorus, percentage of urban land, percentage of rural land, and percentage of forest land) differed significantly among the three regions and a degradation gradient was observed in the NR–MR–SR direction; (3) Canonical correspondence analysis (CCA) revealed that NR sites were characterized by steep slope and coarse substrate, MR sites were characterized by high water temperatures and shallow slopes, and SR sites were primarily characterized by high total phosphorus and ammoniacal nitrogen concentrations; and (4) the Indicator Species Analysis, in conjunction with CCA analysis indicated that the most representative indicator taxon is Tipulidae for NR, Semisulcospira sp. for MR, and Branchiura sp. for SR.