Sphaerorrhiza, a genus of herbaceous plants endemic to the Cerrado domain in Brazil, was recently segregated from the genus Gloxinia and placed in its own monotypic subtribe Sphaerorrhizinae. Most information on this little-known taxon derived from the observation of a single species and a limited number of collections. Using new material representative of all recognized species we reassess the monophyly of Sphaerorrhiza and its phylogenetic placement within Gesnerioideae based on the analysis of nuclear (ITS) and plastid (atpB-rbcL, matK, rpl16, rps16, trnL-trnF) DNA sequences. Our results support the monophyly of the genus and its segregated position from the other clades of Gesneriaceae. A taxonomic revision of Sphaerorrhiza is presented, including new anatomical and morphological data and the first reported chromosome number for the genus. Four species are recognized and two species are described as new, S. rosulata and S. serrata. Descriptions, illustrations, and a distribution map, as well as a key to the species of Sphaerorrhiza, are provided.