The family Plectosphaerellaceae (Glomerellales, Sordariomycetes) includes numerous plant pathogenic genera and soil-borne fungal species. Ten genera are currently accepted, including several taxa that occupy an unresolved position within the family. To address this issue, a multilocus sequence analysis was carried out using partial gene sequences from the 28S large subunit nrRNA gene (LSU), the internal transcribed spacer (ITS) regions of the nrDNA region, including the 5.8S nrRNA gene, the translation elongation factor 1-alpha (TEF1-α), tryptophan synthase (TS), actin (ACT) and the RNA polymerase II second largest subunit (RPB2), based on a large set of isolates mainly from the CBS collection. Results of the molecular data combined with a detailed morphological study resolved 22 genera in the family, of which 12 are newly described. Additionally, 15 new species and 10 new combinations are proposed. An epitype and neotype are also introduced for Stachylidium bicolor and Plectosphaerella cucumerina, respectively.