The Didymellaceae is one of the most species-rich families in the fungal kingdom, and includes species that inhabit a wide range of ecosystems. The taxonomy of Didymellaceae has recently been revised on the basis of multi-locus DNA sequence data. In the present study, we investigated 108 Didymellaceae isolates newly obtained from 40 host plant species in 27 plant families, and various substrates from caves, including air, water and carbonatite, originating from Argentina, Australia, Canada, China, Hungary, Israel, Italy, Japan, South Africa, the Netherlands, the USA and former Yugoslavia. Among these, 68 isolates representing 32 new taxa are recognised based on the multi-locus phylogeny using sequences of LSU, ITS, rpb2 and tub2, and morphological differences. Within the Didymellaceae, five genera appeared to be limited to specific host families, with other genera having broader host ranges. In total 19 genera are recognised in the family, with Heracleicola being reduced to synonymy under Ascochyta. This study has significantly improved our understanding on the distribution and biodiversity of Didymellaceae, although the placement of several genera still need to be clarified.