Penicillium species belong to main causative agents of food spoilage leading to significant economic losses and potential health risk for consumers. These fungi have been isolated from various food matrices, including table eggs. In this study, both conventional Polymerase Chain Reaction (PCR) and Polymerase Chain Reaction-Internal Transcribed Spacer-Restriction Fragment Length Polymorphism (PCR-ITS-RFLP) methods were used for species identification of Penicillium (P.) spp. isolated from the eggshells of moldy chicken eggs. Seven restriction endonucleases (Bsp1286I, XmaI, HaeIII, HinfI, MseI, SfcI, Hpy188I) were applied to create ribosomal restriction patterns of amplified ITS regions. To identify P. verrucosum, P. commune, and P. crustosum with the help of conventional PCR assay, species-specific primer pairs VERF/VERR, COMF/COMR, and CRUF/CRUR were designed on the base of 5.8 subunit-Internal Transcribed Spacer (5.8S-ITS) region. Altogether, 121 strains of microscopic filamentous fungi were isolated by traditional culture mycological examination. After morphological evaluation of both macroscopic and microscopic features, 96 strains were classified in Penicillium spp. Two molecular methods used have confirmed eight isolates as P. verrucosum, 42 isolates as P. commune, and 19 isolates as P. crustosum. Both PCR-ITS-RFLP and conventional PCR assays appear to be suitable alternatives for rapid identification of the above mentioned Penicillium species.