To investigate the genetic diversity of Pinus tabulaeformis distributed in different altitude regions and explore the association of P. tabulaeformis with the environment, the relationship between genetic diversity and environmental factors were analyzed. Inter Simple Sequence Repeat (ISSR) technique was used to evaluate the genetic diversity and population structure of 118 individuals from four natural populations of P. tabulaeformis at different altitudes in Liaoheyuan National Forest Park, Hebei Province, China. Thirteen random primers produced a total of 177 securable amplified fragments. The genetic diversity of P. tabulaeformis population level and species level was PPL=60.2775%, h=0.2171, I=0.3222, and PPL=98.33%, h=0.38142, I=0.5550, respectively. The level of genetic variation varied regularly with different altitudes. The highest level of genetic variation of the population was observed at the altitude of 1354-1274 m (Pop2), and it declined for the populations at the altitude of 1484-1430 m (Pop2), 1129-1091 m (Pop3), and 1002-901 m (Pop4). The high value of differentiation (Gst=0.6562) and analysis of molecular variance (AMOVA) indicated there was significant genetic differentiation among populations, and about two-five of the variation occurred among populations. Pearson correlation analysis further revealed that the genetic diversity within populations was significantly correlated with the altitude and soil nutrient factors (i.e., organic matter, available phosphorus, and available phosphorus). Mantel tests showed that there existed a correlation between the genetic distances among populations and the distance of elevation, and the divergence of soil nutrient factors. The results of the present study suggested that the genetic differentiation among populations of P. tabulaeformis from different altitudes may be caused by the ecological factors of different altitudes and gene flow.