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This study aimed to investigate and identify the isolated five species of Penicillium from different rot fruits. The P.implicatum, P.aurantiogriseum, P.purpurogenum, P.citrinum, and P.notatum Penicillium species were found. Penicillium species are important in the field of medicine, industry, and produces secondary metabolite, some penicillium species are contaminant for bread, grains, fruits, and processed food, so the identification and characterization of these species are very important. By using the microscopic technique and spectroscopic methods for identification and characterization that species isolated from infected fruits. The spectroscopic methods are Raman spectroscopy and UV-Visible spectrophotometry, they are fast analysis techniques, sensitive, use a small quantity of sample, and depend on biochemical components for the discriminate of Penicillium spp. Raman vibrational spectra and UV-Visible absorption spectra showed differences for five Penicillium species. Our results proved that spectroscopic techniques showed spectral differences for five Penicillium species, and this agreed with the study of morphological characteristics for that species. The conclusions of this study demonstrate that spectroscopic methods in combination with morphological techniques could be accurate and reliable approach for discrimination between different Penicillium species.