Medicago rugosa and M. scutellata, two annual species of the genus Medicago, have aroused considerable interest because they carry useful traits that could be introduced into alfalfa and have a chromosome number (2n = 30) that is quite unusual in Medicago. A cytogenetic and molecular study was undertaken to investigate the annual diploid species with 2n = 16 and 2n = 14 that seem to be the most closely related to M. rugosa and M. scutellata, with the aim of characterizing these diploid species and determining their genetic relationship with the species with 2n = 30. Karyological analysis established that some of the diploid species investigated were more similar than the others to both M. rugosa and M. scutellata (as in the case of M. intertexta, M. rotata, and M. polymorpha) or at least to one of those two species (as was the case with M. doliata, M. muricoleptis, and M. murex). RFLP analysis identified four species, namely M. intertexta and M. muricoleptis with 2n = 16, and M. polymorpha and M. murex with 2n = 14, as having the highest degree of genetic affinity with the two species with 30 chromosomes. These findings suggest the possibility of identifying the ancestors of M. rugosa and M. scutellata among those four species and therefore of verifying the probable allopolyploid origin of the two species in question. Keywords: Medicago, annual species, karyotypes, RFLPs.