Based on the significant difference of morphology, chromosome karyotype analysis were carried out in Medicago sativa L.,Medicago falcata L. and alfalfa with cream flower. The results showed: three genotypes of materials had unambiguous difference in chromosome karyotype due to the satellite chromosomes in alfalfa with cream flower and Medicago falcata L.which included 2 terminal point chromosomes. The karyotype formula of Medicago sativa L.,Medicago falcata L. and alfalfa with cream flower respectively were: 2n=4x=24m+8sm，2n = 4x = 24m + 6sm +2T(SAT) and 2n=4x=24m+8sm (SAT). The chromosome similarity in Medicago sativa L. and alfalfa with cream flower was 89% and the dissimilarity was owing to the satellite condition in No.7 of Medicago sativa L. and No.15 of alfalfa with cream flower. The chromosome of Medicago falcata L. was very unique which constructed of Medicago sativa L. and alfalfa with cream flower from chromosome No.19. No.15 chromosome in alfalfa with cream flower had the higher similarity with No.25 chromosome in Medicago falcata L. and No.7 chromosome in Medicago sativa L.,but there were still difference in satellite condition.