This study is the first comprehensive survey of the distribution of freshwater sponges in the rivers of Estonia. The classical morphological method for the identification of freshwater sponges was complemented by a molecular approach, based on sequencing of a DNA region containing a variable D3 domain of the 28S rDNA. This particular segment of 28S RNA proved to be suitable for the discrimination between freshwater sponges at the species level. We showed that Ephydatia fluviatilis was the most widely distributed species followed by Spongilla lacustris and Ephydatia muelleri. Eunapius fragilis was found in a small number of localities and has been described for the first time in Estonia. © E. Schweizerbart'sche Verlagsbuchhandlung 2007.