The genetic diversity of twenty four populations of Medicago ruthenica distributed in Mongolian Plateau and Daxing’an mountain region was analyzed using simple sequence repeat (SSR) markers.This work aimed to know the genetic variation patterns within and among populations and the relationship between population distribution and environments.(1)10 primers were used and five of them showed polymorphism.The results indicated that 5 loci totally have 15 variation alleles,and the average polymorphic information content(PIC) was 0.342 3.(2)The variation among populations was lower which showed by gene differentiation coefficient (Fst=0.17) .There were 82.8% of the variation in M.ruthenica within population，the gene flow （Nm） was 1.2.(3)The cluster analysis indicated the 24 populations could be divided into three distinct groups.The population in northern of Daqinshan (hs) was the single population group.The populations in east,southeast and northeast of Inner Mongolia（kh,hb1,hb2,hb3,lq,lx,bl,zs,lh） were assembled as the second group and the others from northeast of Mongolia plateau transition to the Daxinganling region(dm,db,xs,eg,xb,eh,al,kj,aq1,dw,hb4,ys,aq2,xl) were assembled as the third group.(4)The Mantel’s test of the relationship between genetic distance and the environment factors showed that the genetic distance among M.ruthenica was affected by altitude,latitude and longitude.The impacts of geographical location (latitude and longitude) was greater than altitude and the single latitude or longitude（r=0.402 7,P<0.01）.Between latitude and longitude,the impact of longitude was larger.