Abstract: Conidia are asexual reproduction cells of Penicillium, and their karyotype characteristics play an important role in the growth, reproduction, genetics and breeding of Penicillium. The conditions for achieving the highest proportion of uninucleate conidiospores are being sought by using water agar and Czapek–Dox medium over different cultivation periods. The karyotype characteristics of conidiospores of Penicillium griseofulvum are observed under fluorescence microscopy using DAPI staining, with observations being conducted under water agar and Czapek–Dox medium conditions over different time periods. The findings revealed that during the growth and development of Penicillium griseofulvum conidia, mononuclear, binucleate, and multinucleate phenomena were concurrently observed, with diverse karyotypes. The proportions of various karyotypes among conidia in different media exhibited significant differences（P＜0.001）. In the same medium, the proportion of conidia with a specific karyotype remained relatively stable over different culture times, showing no significant difference（P＞0.05）. These results provide a theoretical reference for better genetic manipulation of Penicillium griseofulvum.