In order to clarify the relationships (genetic exchange and shared ancestry) between natural and cultivated populations of alfalfa (Medicago sativa L.) in Spain, we investigated the patterns of mitochondrial DNA variation (characterized through restriction fragment length polymorphism) for 248 individuals in seven natural and six cultivated populations of this species. Mitochondrial variation was evidenced in both natural and cultivated populations of M. sativa. Among the seven mitotypes idengified in the species, two were specific of the natural populations, a result attesting the fact that the Spanish wild form of M. sativa is an original genetic pool compared to the cultivated one. Other mitotypes were observed in both natural and cultivated populations, suggesting the occurrence of gene flow through seeds from cultivated towards natural populations. Comparisons with previously gathered nuclear and phenotypic data give insights into the different evolutionary forces acting on the different kinds of Spanish natural populations examined so far.