The Steelpoort River is affected by a wide range of pollutants resulting from mining, industrial and agricultural activities and domestic sewage. Benthic macroinvertebrates are often used to assess water quality as they respond to changes in the aquatic environment. The study was to assess the benthic macroinvertebrate communities in the Steelpoort River along a pollution gradient, from less polluted upstream to highly polluted downstream. The impacted downstream sites generally had the highest turbidity, total dissolved solids (TDS) and conductivity values but lowest dissolved oxygen. While the upstream sites generally exhibited low turbidity, TDS and conductivity values but high dissolved oxygen. The physical and chemical changes between sites were accompanied by shifts in macroinvertebrate community composition. The sensitive macroinvertebrate families such as Baetidae, Leptophlebiidae and Perlidae dominated the upstream sites with low water turbidity, low conductivity, low TDS, and high dissolved oxygen while the tolerant families such as Chironomidae, were abundant in highly impacted downstream sites with high water turbidity, high conductivity, high TDS, and low dissolved oxygen. The study revealed differences in physico-chemical parameters, and in the composition of the macroinvertebrate community. Hence, changes in the abundance and diversity of macroinvertebrates reflected the longitudinal pollution gradient in the river.