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In North African oases Medicago sativa ssp. sativa is likely to have been cultivated for long time without contacts with ssp. falcata. In order (i) to verify the genetic distinctiveness of the alfalfa germplasm of the oasis and (ii) to examine their internal variation, 17 farm landraces were collected in Siwa oasis (Egypt) and studied with four Egyptian and three Italian cultivars (120 plants/population) by means of bio-agronomic characters in 2 years cultivation and SSR markers in ’bulk’ samples. Siwa germplasm was similar to the cultivars for dry matter yield but differed for plant form, earliness and reaction to fall conditions; SSR analysis based on 31 polymorphic DNA fragments distinguished Siwa landraces from the two groups of varieties. The variation within Siwa group was of continuous type based on bio-agronomic traits; average genetic similarity was high (0.8959) and UPGMA dendrogram suggests poor isolation among the landraces, although all, except two, were uniquely fingerprinted. The use of putative ’pure’ subspecies of high agronomic value in the alfalfa variety construction process is discussed.