We related macroinvertebrate community structure to physical, chemical, and biological gradients in flow-through constructed wetlands receiving secondarily treated domestic wastewater and lower-nutrient river water in Ohio, USA. Macroinvertebrates collected with benthic colonization plates and emergence traps were analyzed with diversity, biotic, and combination indices and related to seventeen parameters of water quality, substrate characteristics, and primary productivity in both wetland systems. A total of 36 and 39 macroinvertebrate taxa were collected in the wastewater wetland and river wetland, respectively. Invertebrate communities nearest to the wastewater source in the wastewater wetland had significantly lower (mean ± SE) Simpson diversity (0.5 ± 0.13) and Invertebrate Community Index (ICI) scores (2.6 ± 0.8) than those of other sampling locations. Sampling locations near the middle and outflow of the wastewater wetland had diversity scores (0.84 ± 0.01) and ICI scores (4.8 ± 0.7) statistically similar to sites in the river-fed wetlands. Average diel dissolved oxygen and specific conductivity were the best environmental predictors of invertebrate community metrics; these variables along with chemical oxygen demand and nitrate-nitrogen described nearly 90% of the ICI variation in a four-predictor regression model. Multiple correspondence analysis showed sampling locations loosely grouped by average diel dissolved oxygen, which described 65.4% of the overall variation in the gradient data, and specific conductivity, which described and additional 16.4%. Macroinvertebrate analyses are presented as a valuable means of assessing the ecological status of wetlands used to improve water quality and as an important supplement for traditional chemical analyses.