Analyzing the ploidy levels of plants is important for identifying species, selecting parental lines, identifying the relationships between species, and determining evolutionary patterns. The genus Chrysanthemum is widely distributed throughout the world and exhibits different ploidy levels. We used flow cytometry to analyze the ploidy levels of nine species of Chrysanthemum L. collected from different regions and geographical locations in China. Three diploids from Henan and Wuhan provinces corresponded to Chrysanthemum lavandulifolium and two species of C. nankingense,while three tetraploids from various regions corresponded to C. indicum and two species of C. chanetii. Two hexaploids corresponding to C. vestitum were collected at Funiu mountain(Henan province), and C. zawadskii was collected at Huangshan mountain(Anhui province). We found that OTTO extraction buffer was suitable for extracting nuclei from most species, apart from C. zawadskii. Flow cytometry proved to be a simple, rapid, and highly accurate method for identifying ploidy levels in Chrysanthemum species. Analyzing the ploidy levels of plants is important for identifying species, selecting parental lines, identifying the relationships between species, and determining evolutionary patterns. The genus Chrysanthemum is widely distributed throughout the world and exhibits different ploidy levels. We used flow cytometry to analyze the ploidy Three of the nine species of Chrysanthemum L. collected from different regions and geographical locations in China. Three diploids from Henan and Wuhan were corresponded to Chrysanthemum lavandulifolium and two species of C. nankingense, while three tetraploids from various regions corresponded to C. indicum and two species of C. chanetii. Two hexaploids corresponding to C. vestitum were collected at Funiu mountain (Henan province), and C. zawadskii was collected at Huangshan mountain (Anhui province). We found that OTTO extraction buffer was suitable for extracting nuclei from most species, apart from C. zawadskii. Flow cytometry proved to be a simple, r apid, and highly accurate method for identifying ploidy levels in Chrysanthemum species.