Background Simao pine ( Pinus kesiya Royle ex Gordon var. langbianensis (A. Chev.) Gaussen) is one of the most important tree species in the production of timber and resin in China. However, the genetic diversity of the natural populations has not been assessed to date. In this study, sequence related amplified polymorphism (SRAP) markers were used to investigate the genetic composition of natural Simao pine populations. Method The SRAP markers were applied and their efficiency was compared using various statistical multivariate methods, including analysis molecular of variance (AMOVA), the unweighted pair group method with arithmetic mean (UPGMA), and Principal coordinate analysis (PCoA). Results The 11 populations revealed a high level of genetic diversity (PPB = 95.45%, H = 0.4567, I = 0.6484) at the species level. A moderately low level of genetic differentiation ( G st = 0.1701), and a slightly high level of gene flow ( N m = 2.4403) were observed among populations using AMOVA. Eleven populations of Simao pine were gathered into four distinct clusters based on molecular data, and the results of UPGMA and PCoA also illustrated that assignment of populations is not completely consistent with geographic origin. The Mantel test revealed there was no significant correlation between geographic and genetic distance ( r = 0.241, p = 0.090). Discussion The SRAP markers were very effective in the assessment of genetic diversity in Simao pine. Simao pine populations display high levels of genetic diversity and low or moderate levels of genetic differentiation due to frequent gene exchange among populations. The low genetic differentiation among populations implied that conservation efforts should aim to preserve all remaining natural populations of this species. The information derived from this study is useful when identifying populations and categorizing their population origins, making possible the design of long term management program such as genetic improvement by selective breeding.