The chromosome flaking of root-tip on germinate seed in Wide Medicago sativa L. can be made clearly, which pretreatmented by the mixture of 0.15% colchicine and 0.002mol/L 8-hydroxyquinoline for 3h, and then, enzymolysissed by 1% cellulase+1% pectinase for 1.5h at 25℃. The result of karyotype analysis shows that the total of chromosome in Wild Medicago sativa L. is 32 with a pair of satellites. The karyotype in such specie is 2B type and the karyotype formulation is 2n=32=30m(2SAT)+2sm, the length construction is 2n=2L+18M2+8M1+4S.