The Mediterranean Basin is a major region of lucerne (Medicago sativa) cultivation. We used seven microsatellite markers to evaluate within-population diversity and test differentiation among lucerne populations from the Mediterranean Basin. A set of 16 populations from France, Italy, Tunisia, Algeria, Morocco, Australia and the USA, each represented by 30 individuals, was analysed for seven microsatellite markers. One marker was excluded from the analyses because of presence of null alleles. Number of alleles and expected heterozygozity were calculated to evaluate within-population diversity. F(IS) was calculated to test departure of populations from panmictic equilibrium. Population structure was analysed by the calculation of F(ST) index. Within-population genetic diversity was large in all populations, and the populations appeared to be at panmictic equilibrium. Global F(ST) was significant but very low (0.016). Three groups were identified, one composed by only Gabes, a second group composed by three North African populations, the two Australian cultivars, one American cultivar and one European population and a third group with six European populations, one Moroccan population and one American cultivar. Differentiation among populations was limited, which could be explained by the numerous possibilities of gene flow among populations, such as seed exchange or pollen flux. Reproductive biology (allogamy) and genetics (autotetraploidy) favour the maintenance of large within-population diversity and limits the possibility of population differentiation for neutral markers. This relative lack of differentiation with neutral markers is observed while the populations are known to noticeably differ for agronomic traits.