Abstract: In order to explore the genetic variation among Pinus bungeana populations, their genetic diversity and differentiation level were studied in 21 natural populations by EST-SSR markers. 14 polymorphic loci were detected in 663 individuals among 21 natural populations using 7 pairs of EST-SSR primes. The effective number of alleles (Ne), Shannon's information index (I), observed heterozygosity (Ho), expected heterozygosity (He) and Nei's expected heterozygosity (Nei's) were 1.156 5-1.601 9, 0.133 5-0.492 5, 0.138 4-0.397 3, 0.086-0.342 8, and 0.084 6-0.337 4 respectively. The results of genetic differentiation analysis showed that the average Fst and Nm were 0.215 2 and 0.911 9 respectively, the gene flow was less and the genetic differentiation was high among populations. Central regions of genetic diversity are mainly in west and south of the distribution and the genetic diversity in distribution changed regularly from west to east and from south to north.