The thick-shelled river mussel, Unio crassus Philipson, 1788, is considered to be one of the species with the highest conservation priority in Serbia. The study represents the first comprehensive research of the distribution of U. crassus in Serbian waters. The research covered a variety of waterbody types throughout Serbia, and distribution data were considered over three time periods from 1953 to 2019. The paper summarizes all the available literature data, field research and information obtained during the review of the collection of malacological material of the Natural History Museum in Belgrade. The results show a positive population trend, which is reflected in an extension of the distribution area and an increase in population density. After reviewing the museum collection, 13 synonyms for U. crassus were identified. The study also revealed a better insight into the habitat requirements and the limiting factors of the species. Substrate characteristics, waterbody types, altitude, and nitrate content of the water seem to be of great importance for the occurrence of the species. The results presented here can improve further measures for the conservation of U. crassus, not only in Serbia, but also in the Western Balkans.