In this study, we studied the diversity of Penicillium occurring in soil collected along the Woodville Big Tree Forest Trail situated close to the coastal town of Wilderness in South Africa. Strains were accessioned into a collection and then identified to species based on β-tubulin DNA sequences, which is the recommended DNA barcode for the genus. The 74 strains were found to represent 18 species, including six we consider undescribed. Here, we introduce them as Penicillium claroviride, P. kalander, P. mattheeae, P. outeniquaense, P. subfuscum, and P. umkhoba. Phylogenetic comparisons were made, and genealogical concordance was demonstrated for these new species using DNA sequences from nuc rDNA internal transcribed spacer region ITS1-5.8S-ITS2 (ITS barcode), β-tubulin, calmodulin, and RNA polymerase II second largest subunit. Notes on morphological characters distinguishing the new species from their close relatives are provided. [ABSTRACT FROM AUTHOR]