A major flooding event that occurred during October-November 2012 caused major changes in the Kowie River hydromorphology and aquatic communities. The aim of our study was to identify the environmental variables that structure riverine benthic diatom communities at upstream and downstream locations 25 km apart on the Kowie River, South Africa. This was undertaken using tiles as artificial substrates so that we could study how the communities developed after the flood disturbance. The diatom community structure was assessed over a 28-day period following a flood event in October 2012. The Mann Whitney test indicated that there was a statistically significant difference (p < 0.05) in total dissolved solids, salinity, pH and oxygen reduction potential between the two sites. In total, 58 diatom species belonging to 30 genera were identified over the 28-day study. Achnanthidium minutissimum, Fragilaria biceps, F. ulna var. acus, Pinnularia borealis and P. acrosphaeria were the most numerically dominant on Day 7 and were considered as early colonisers, while on Day 28, Achnathidium minutissimum, F. capucina, Craticula buderi, C. vixnegligenda, Diploneis subovalis and Gomphonema venusta, the late colonisers, were dominant. The species richness increased from 13 (upstream location) on Day 7 to 22 (both locations) by Day 21. A redundancy analysis showed that total suspended solids, salinity, resistivity, pH and oxygen reduction potential were the most significant physico-chemical variables explaining diatom composition. The results from this relatively smallscale tile experiment indicate the complexity of freshwater benthic diatom community structure and development.