In the present study, nine terverticillate Penicillium isolates (P. griseofulfum, P. puberulum, P. crustosum, P. aurantiogriseum, P. chrysogenum, P. primulinum, P. expansum, P. viridicatum, Eupenicillium egyptiacum) from 56 soil samples were characterized genetically by a PCR method. The DNAs of the strains were isolated using the glass beads and vortexing extraction method and then used for PCR amplification with the internal transcribed spacer 1 (ITS1) and ITS4 universal fungal specific primers. The ITS regions of fungal ribosomal DNA (rDNA) were sequenced through the CEQ 8000 Genetic Analysis System. ITS-5.8S sequences obtained were compared with those deposited in the GenBank Database. The results indicated that the identification of Penicillium species with PCR based methods provided significant information about the solution to taxonomy and improve food safety and to protect the users from harmful contaminants such as mycotoxins, which must be controlled during the production of agricultural materials as well as during the processing of food and feed.