Septoria is a large genus of asexual morphs of Ascomycota causing leaf spot diseases of many cultivated and wild plants. Host specificity has long been a decisive criterium in species delimitation in Septoria, mainly because of the paucity of useful morphological characters and the high level of variation therein. This study aimed at improving the species delimitation of Septoria by adopting a polyphasic approach, including multilocus DNA sequencing and morphological analyses on the natural substrate and in culture. To this end 365 cultures preserved in CBS, Utrecht, The Netherlands, among which many new isolates obtained from fresh field specimens were sequenced. Herbarium material including many types was also studied. Full descriptions of the morphology in planta and in vitro are provided for 57 species. DNA sequences were generated for seven loci, viz. nuclear ITS and (partial) LSU ribosomal RNA genes, RPB2, actin, calmodulin, Btub, and EF. The robust phylogeny inferred showed that the septoria-like fungi are distributed over three main clades, establishing the genera Septoria s. str., Sphaerulina, and Caryophylloseptoria gen. nov. Nine new combinations and one species, Sphaerulina tirolensis sp. nov. were proposed. It is demonstrated that some species have wider host ranges than expected, including hosts from more than one family. Septoria protearum, previously only associated with Proteaceae was found to be also associated with host plants from six additional families of phanerogams and cryptogams. To our knowledge this is the first study to provide DNA-based evidence that multiple family-associations occur for a single species in Septoria. The distribution of host families over the phylogenetic tree showed a highly dispersed pattern for 10 host plant families, providing new insight into the evolution of these fungi. It is concluded that trans-family host jumping is a major force driving the evolution of Septoria and Sphaerulina.