Penicillium species are ubiquitous in all kinds of environments, and they are of industrial, agricultural and clinical importance. In this study, soil fungal diversity in Southwestern China was investigated, and that of Penicillium turned out to be unexpectedly high. The survey included a total of 179 cultures of the genus isolated from 33 soil samples. Three-locus phylogenetic analyses and morphological comparisons were carried out. The examinations revealed that they belonged to two subgenera (Aspergilloides and Penicillium), 11 sections (Aspergilloides, Canescentia, Citrina, Exilicaulis, Fasciculata, Gracilenta, Lanata-Divaricata, Penicillium, Ramosum, Robsamsonia, and Sclerotiorum), 25 series, and 74 species. Forty-three species were discovered as new to science, and a new series, Simianshanica, was established in sect. Aspergilloides. Additionally, 11 species were recorded for the first time in China. Species isolation frequency and distribution of the group were also discussed.