Diaporthe (Phomopsis) species have often been reported as plant pathogens, non-pathogenic endophytes or saprobes, commonly isolated from a wide range of hosts. The primary aim of the present study was to resolve the taxonomy and phylogeny of a large collection of Diaporthe species occurring on diverse hosts, either as pathogens, saprobes, or as harmless endophytes. In the present study we investigated 243 isolates using multilocus DNA sequence data. Analyses of the rDNA internal transcribed spacer (ITS1, 5.8S, ITS2) region, and partial translation elongation factor 1-alpha (TEF1), beta-tubulin (TUB), histone H3 (HIS) and calmodulin (CAL) genes resolved 95 clades. Fifteen new species are described, namely Diaporthe arengae, D. brasiliensis, D. endophytica, D. hongkongensis, D. inconspicua, D. infecunda, D. mayteni, D. neoarctii, D. oxe, D. paranensis, D. pseudomangiferae, D. pseudophoenicicola, D. raonikayaporum, D. schini and D. terebinthifolii. A further 14 new combinations are introduced in Diaporthe, and D. anacardii is epitypified. Although species of Diaporthe have in the past chiefly been distinguished based on host association, results of this study confirm several taxa to have wide host ranges, suggesting that they move freely among hosts, frequently co-colonising diseased or dead tissue. In contrast, some plant pathogenic and endophytic taxa appear to be strictly host specific. Given this diverse ecological behaviour among members of Diaporthe, future species descriptions lacking molecular data (at least ITS and HIS or TUB) should be strongly discouraged.