The present study aimed to characterize the chromosome features of cherry (Prunus pseudocerasus Lindl.) germplasm in Guizhou Province, China, in order to facilitate the selection and breeding programs of this economically and ornamentally valuable species. The stem tip chromosome preparation technique was employed for ploidy identification and karyotype analysis, and the results were further validated by flow cytometry. The findings revealed that the 28 cherry accessions from Guizhou province exhibited a chromosomal base of x = 8, comprising 19 tetraploid and nine hexaploid individuals. Karyotype analysis showed two chromosome types, “m” and “sm,” with the longest chromosome/shortest chromosome (Lc/Sc) ranging from 1.6 to 2.65, the mean arm ratio (MAR) varying from 1.15 to 1.56, and index of the karyotypic asymmetry (As.K) ranging from 53.74 to 61.6. Three karyotype types, “1A,” “1B,” and “2B,” were identified among the studied accessions. The most evolutionarily advanced accession was HZ152, while DCZC27 represented the most primitive karyotype. This study expands the ploidy database of Chinese cherry and provides valuable information for the conservation and utilization of cherry germplasm resources in Guizhou province.