Abstract The subfamily Sphaeriinae is a diverse and ecologically significant group of freshwater invertebrates, playing a keystone role in the energy flow and nutrient cycling in aquatic ecosystems. Despite a long history of its study, the taxonomic structure and species content of this group are a matter of ongoing disputes between malacologists. We clarify the taxonomy, distribution, and evolutionary biogeography of the Sphaeriinae around the world based on the largest dataset of DNA sequences of these clams ever accumulated, which includes the data on specimens collected from various continents and countries around the globe. Our 16S rRNA and two-locus phylogenies reveal that the Sphaeriinae is a monophyletic group, containing at least seven genus-level clades: Sphaerium Scopoli, 1777, Pisidium C. Pfeifffer, 1821 (s.s.), Euglesa Jenyns, 1832, Odhneripisidium Kuiper, 1962, Conventus Pirogov & Starobogatov stat. nov., 1974, ‘Pisidium’ sterkianum group, and Hindupisidium Vinarski & Bespalaya gen. nov. Our 16S rRNA phylogenetic tree contains 80 well-supported clades corresponding to the putative species-level taxa. Four species new to science are described here: Euglesa kolymensis Bespalaya, Aksenova & Bolotovsp. nov., Euglesa kyrgyzica Bespalaya, Aksenova, Kondakov & Palatovsp. nov., Euglesa moroccana Bespalaya, Vinarski, Aksenova, Mabrouki, Kondakov & Palatovsp. nov., Odhneripisidium caucasus Bespalaya, Vinarski, Aksenova, Kondakov & Palatovsp. nov. Our fossil-calibrated phylogeny and generalized biogeographic model indicate that the most recent common ancestor of the Sphaeriidae could have originated somewhere within a broad area covering China near the Lower Cretaceous boundary.