The phylogeny of Trichoderma and the phylogenetic relationships of its species was investigated by maximum parsimony analysis and distance analysis of DNA sequences from multiple genetic loci. 18S rDNA sequence analysis suggests that the genus Trichoderma evolved at the same time as Hypomyces and Fusarium and thus about 110 Myr ago. 28S rDNA sequence analysis shows that the genus Trichoderma is part of a monophyletic branch within the Hypocreaceae, which also includes Arachnocrea and Aphysiostroma in basal positions. Gene trees inferred from a combined analysis of the nuclear ribosomal internal transcribed spacer (ITS1 and 2), the D1 and D2 region of the 28S rDNA, the small subunit of the mitochondrial rDNA (mitSSU), the fifth and part of the sixth exon of translation elongation factor 2 (tef1), and a fragment of ech42 provide strong statistical support for a phylogeny consistent with the existence of four clades: clade A comprises species of Bissett's (1991) sect. Trichoderma but also T. hamatum, T. pubescens, T. asperellum, and T. strigosum; clade C comprises all the species contained in section Longibrachiatum as revised by Samuels et al. (1998), and clade D contains only T. aureoviride which is genetically most distant to all other species. Clade B, on the other hand, contains a large and taxonomically heterogeneous mixture of species, among which several subclades could be identified: subclade B1 containing H. lactea, H. citrina, H. citrina var. americana, H. lutea, and an unnamed T. sp. 1; subclade B2 containing T. stromaticum, and an unnamed T. sp. PPRI3559; subclade B3 containing T. fertile, T. oblongisporum, and H. hunua; subclade B5 containing T. polysporum, T. croceum, Hypocrea pilulifera, and T. minutisporum; and a larger subclade (B4), in which three strain clusters could be distinguished: one comprising T. harzianum, T. inhamatum, H. vinosa, and T. aggressivum, another one containing T. fasciculatum, T. longipile, and T. strictipile; and a third containing T. virens, T. flavofuscum, and T. crassum. The position of the remaining species of subclade B4 (T. spirale, T. cfr aureoviride, H. tawa, and T. tomentosum) was not resolved. A comparison of the topologies of the individual gene trees was concordant with the topology of the combined tree in most cases, but also revealed incongruent positions for a few species (T. oblongisporum, T. longipile, T. fasciculatum) which was most pronounced in the ITS1 and 2 tree. The results confirm the recent concept for sects Longibrachiatum and Trichoderma, indicate that the sects Hypocreanum and Pachybasium cannot be distinguished phylogenetically, and provide a first phylogenetic basis for dissection of the latter two sections.