Background. The subfamily Sphaeriinae of freshwater bivalves is formed by three genera, Musculium, Pisidium,and Sphaerium. However, the monophyly of the latter two has been questioned in different phylogenetic analyses. Goals. In this study, we added Pisidiumchilenseto the phylogenetic analysis, based on sequences of nuclear ribosomal, an internal transcribed spacer(ITS-1). Methods.The data matrix was analyzed with maximum parsimony and Bayesian inference. Results support the monophyly of Sphaeriinae. The genera Pisidium and Musculium are monophyletic, while Sphaerium is paraphyletic with respect to Musculium. Within Pisidium, the South American species and sole representatives included in this analysis of the subgenus Afropisidium formed a monophyletic group. Conclusions. Before advancing new biogeographic or taxonomic scenarios for Sphaeriinae, further analyses based on denser taxonomic and character sampling are needed.