The Sphaeriidae represent one of the primary molluscan radiations into freshwater environments. Wehave reconstructed phylogenetic relationships of the Sphaeriinae, a cosmopolitan sphaeriid subfamily, using variation in nuclear ribosomal first internal transcribed spacer (ITS-1) and mitochondrial large ribosomal subunit (16S) gene fragments. A total of 38 New World, Eurasian and Antipodean taxa were characterized, including all primary taxonomic groupings except for Neopisidium, and members of the sister clade Euperinae were employed as outgroups. Phylogenetic analyses of individual and combined (16S + ITS1) datasets all recovered a paraphyletic Pisidium and a derived clade of asynchronous brooding Sphaerium/Musculium taxa. Maximum parsimony as well as maximum likelihood analyses of combined data yielded largely congruent and well-resolved topologies, and robustly supported clades were utilized to revise current sphaeriine taxonomy. Instead of the traditionally accepted three cosmopolitan genera, Pisidium s.l., Musculium, and Sphaerium, five major monophyletic lineages, Afropisidium, Odhneripisidium, Pisidium, Cyclocalyx, and Sphaerium, were recognized at the generic level. In addition, a number of subgeneric level groups were recovered in Sphaerium: Herringtonium, Sphaerium s.s., Sphaerinova, Amesoda, and Musculium, together with one unassigned species, S. transversum.