Resumo. Abstract Aim: This study aimed to evaluate the influence of seasonal variation in the hydrological regime and of limnological variables on species richness of periphytic algae in lakes of the Upper Paraná River floodplain. Methods The study area is located in the last undammed stretch of the Paraná River in this floodplain, in which three lakes were sampled quarterly (May, August and November 2014, and February and May 2015) for one year. The periphytic material was obtained from petioles of the aquatic macrophyte Eichhornia azurea (Sw.) Kunth. Results We registered 149 taxa of periphytic algae, distributed in 9 classes. Algal richness changed as a function of the seasonal dynamics of the Paraná River’s hydrological regime. Water level fluctuations directly influenced periphytic algae species. During periods of high water level, species richness increased. Limnological variables showed a close relationship with the hydrological regime, and the dissolved nutrients and turbidity were the abiotic factors that most influenced the algal community. The following species were present in all lakes: Fragilaria capucina Desmazières, Gomphonema gracile Ehrenberg and Navicula cryptotenella Lange-Bertalot (Bacillariophyceae) and two species of the genus Oedogonium (Oedogoniophyceae). Conclusions The hydrological regime of the Paraná River played an important role in structuring communities of periphytic algae. Seasonal variation in hydrological regime and in limnological variables together influenced the species richness of these organisms in these floodplain lakes.