The karyomorphology of 18 Epimedium species were reported in this study. Among 18 species, the karyomorphology of eight taxa, namely E. ecalcaratum, E. pauciflorum, E. lishihchenii, E. dolichostemon, E. pudingense, E. baojingense, E. yingjiangense and E. shuichengense were reported for the first time. The chromosome numbers of all species studied are 2n = 2x = 12, except E. yingjiangense 2n = 4x = 24. Tetraploid was reported in genus Epimedium for the first time. All the Epimedium species, investigated in the present research, have a uniform karyotype with two types of chromosome, metacentric and submetacentric, except E. leptorrhizum and E. sagittatum, of which the sixth pair of chromosomes were subteleocentric. A secondary constriction was commonly found on the proximal side of the short arm of the chromosome pair 1 or 2. The karyotype symmetry of all investigated species was relatively primitive 1A, 2A or 3A. The speciation, infrageneric and intergeneric relationship of Epimedium in Berberidaceae were discussed.