Penicillium simplicissimum is a broadly circumscribed taxon often encountered on soil community lists and in biotechnological reports. Phylogenetic analysis of the ITS1- 5.8S-ITS2 rDNA region indicated that P. simplicissimum s.l. includes several species. Recognition of these species is further supported by differences in micromorphological characters, particularly of the conidia and phialides, and the production of distinct profiles of secondary metabolites by each species. Group-I introns, located in the SSU rDNA, were identified in six of the 21 isolates; their presence was used to test hypotheses about the monophyly of the 19 pulvillorum and other intron-bearing strains. Our results show that 19 brasilianum, P. ochrochloron, P. piscarium, P. pulvillorum and P. simplicissimum should be recognized as distinct and valid species. Three additional, undescribed species were identified within the P. simplicissimum group, here referred to as Penicillium spp. I, II and III. The name P. simplicissimum should be used only for isolates which closely match the neotype and NRRL 902 in all characters.