Twenty polymorphic mirosatellite markers have been developed and characterized from RAPD product in Populus euphratica for protection of its natural resources. Polymorphisms of these microsatellite markers were evaluated in a natural population of 30 individuals collected from Xinjiang Region in China. The number of alleles ranged from 3 to 12. Observed and expected heterozygosities varied from 0.53 to 0.97, and from 0.56 to 0.94, respectively. All the microsatellite loci conformed to Hardy–Weinberg equilibrium after Bonferroni correction. They have showed sufficient level of polymorphisms to estimate the population structure, genetic diversity and species conservation. It is an efficient method to isolate microsatellite markers from RAPD product in P. euphratica.