Microsatellite variation of Picea asperata Mast. originating from the mountains of China was investigated by analyzing variation at seven SSR loci in 250 individuals representing ten populations. A fair degree of genetic diversity and considerable population subdivision occurred with the mean gene diversity (H) of 0.707, and genetic distances among populations varying between 0.121 and 0.224 (FST) and between 0.100 and 0.537 (RST). However, inter-population genetic distances showed no correlation with geographic distances between the population sites. This ruled out a simple isolation by distance model and suggested that migration does not have a great impact. In fact, the amount of gene flow, detected using private alleles, was very low, equaling only 0.753. Allele permutation tests revealed that stepwise-like mutations, coupled with genetic drift, could contribute to population differentiation. Moreover, significant genetic differences between populations were detected at most loci. The results indicate that natural selection, presumably through environmental stress, may be one of the main factors causing micro-geographical differentiation in the genetic structure of P. asperata. Based on SSR genotypes, 70% of the 250 individuals were correctly classified into their sites of origin. This suggests that microsatellites (SSRs) are effective in distinguishing genotypes of P. asperata originating from diverse eco-geographical sites in China.