Species of Didymellaceae have a cosmopolitan distribution and are geographically widespread, occurring in diverse ecosystems. The family includes several important plant pathogenic fungi associated with fruit, leaf, stem and root diseases on a wide variety of hosts, as well as endophytic, saprobic and clinically relevant species. The Didymellaceae was recently revised based on morphological and phylogenetic analyses of ex-type strains subjected to DNA sequencing of partial gene data of the LSU, ITS, rpb2 and tub2 loci. Several poly- and paraphyletic genera, including Ascochyta, Didymella and Phoma were redefined, along with the introduction of new genera. In the present study, a global collection of 1 124 Didymellaceae strains from 92 countries, 121 plant families and 55 other substrates, including air, coral, human tissues, house dust, fungi, insects, soil, and water were examined via multi-locus phylogenetic analyses and detailed morphological comparisons, representing the broadest sampling of Didymellaceae to date. Among these, 97 isolates representing seven new genera, 40 new species and 21 new combinations were newly introduced in Didymellaceae. In addition, six epitypes and six neotypes were designated to stabilise the taxonomy and use of older names. A robust, multi-locus reference phylogenetic tree of Didymellaceae was generated. In addition, rpb2 was revealed as the most effective locus for the identification of Didymellaceae at species level, and is proposed as a secondary DNA marker for the family.