Astronautical mutagenesis is a new breeding technique. In space, the variation are produced by means of the comprehensive effect like high vacuum, microgravity, intense radiation and so on. The mutated seeds by astronautical mutagenesis will be selected to breed the new cultivars. Astronautical mutation breeding opens up a new way for breeding. In this study, the genetic variation of 10 “Zhaodong” Alfalfa mutated lines produced by astronautical mutagenesis were investigated using RAPD method. For RAPD analysis, 90 primers were tested and 21 primers were selected for the detailed RAPD makers. Comparing with the original “Zhaodong” Alfalfa, the genetic variation of mutated lines was very obvious. For the same Primer, there were more one band (a few bands) or less one band (a few bands) between mutant and its original lines. The amplified bands and polymorphic sites were rich, ranging from 250-2000bp.1108 RAPD loci were determined. The quantity of RAPD polymorphic loci ranged from 2-31. The rate of polymorphic sites of the mutants and wild types ranged from 2.06%-31.95%. The mutated line-8, 9, 10 had bigger variation and mutated line-10 has the biggest variation in all mutants, it can be for the further selection and breeding work for the new alfalfa.