Altitude is thought to have greatly influenced current species distribution and their genetic diversity. However, it is unclear how different altitudes have affected the distribution and genetic diversity of Picea likiangensis, a dominant forestry species in the Qinghai-Tibetan Plateau region (QTP). In this study, we investigated the genetic diversity of Picea likiangensis populations which distributed in different altitudes of QTP using EST-SSR markers. The results suggested that this species has high genetic diversity at species level, with 100% of loci being polymorphic and an average Nei’s gene diversity (He) of 0.7186 and Shannon’s information index (I) of 1.5415. While the genetic diversity of Picea likiangensis at population level was lower than that at species level, with He and I being 0.6562 and 1.3742, respectively. The variation in genetic diversity of all four studied populations indicated a low-high-low pattern along the elevation gradients. The mid-elevation population (3050 m) was more genetically diverse than both low-elevation (2900 m) and high-elevation populations (3200 m and 3350 m). Nei’s genetic diversity (Fst = 0.0809) and AMOVA analysis (Phist = 0.1135) indicated that a low level of genetic differentiation among populations. Gene flow among populations was 2.8384, suggesting that high gene flow is a main factor leading to high levels of the genetic diversity among populations.