Abstract: The genetic evolution analysis of four mainly restore lines for hybrid rice including Minghui63,Minghui86,Hang1and Hang2with similarly genetic background were carried out in the paper.A total of 134pairs of simple sequence repeats(SSR)were selected distributed evenly from twelve chromosomes of rice(Oryzae sativa L.).The results indicated that there were a lot of polymorphic loci between breeding varieties under space flight mutation(Hang1,Hang2)and original variety(Minghui86),39polymorphic loci with a polymorphic ratio of29.10%.The number of alleles per loci ranged from 1to 4with an average of 2.1.The value of polymorphism information content(PIC)was 0.37.The four mainly restore lines were classified into 2groups by clustering.It is distinction between Hang 1,Hang2and Minghui 86when the threshold value is 0.87.A further analysis on SSR markers distributed in two sides of marker RM234 which have key gene regions,indicated that there were 5polymorphism sites from 7pairs of SSR markers.Bioinformatics analysis showed that those loci have important biological function in Minghui 86,was mutated under space flight mutation.Furthermore,breeding excellent restoring line Hang 1and Hang 2,provided a molecular evidence that advantageous variations are in space flight mutation for rice breeding.