The study was conducted to assess the species composition, substrate specificity, and seasonal abundance of periphytic algae from the river Periyar. Monthly samples were collected for one year (June 2016 – May 2017) from different substrates of five selected stations. Eight physicochemical variables such as temperature, dissolved oxygen, pH, conductivity, chloride, sulfate, nitrate, and phosphate were also monitored during the study. Taxonomic studies recorded 156 species of periphytic algae belonging to 56 genera, 36 families, and 5 classes. Naviculaceae was the most abundant family followed by Fragilariaceae and Pinnulariaceae. The principal component analysis revealed the dominance of periphytic algae in the pre-monsoon period. Canonical correspondence analysis indicates pH, conductivity, and sulfate plays a crucial role in periphytic algal assemblages. Correspondence analysis and percentage abundance among different substrates showed the preference of leaf substrate for primary colonization and subsequent succession. The study signifies the importance of substratum and environmental variables in the dynamics of periphytic algal community composition and abundance.