Genetic diversity and population genetic structure of Shorea leprosula was investigated using seven natural populations distributed throughout Peninsular Malaysia and one natural population from Borneo. The mean population and species level genetic diversity were exceptionally high (He = 0.369 ± 0.025 and 0.406 ± 0.070, respectively). Heterozygosity varied among populations, ranging from 0.326 to 0.400, with the highest values found in the populations from central Peninsular Malaysia. Correlations among ecological factors (longitude, latitude, and annual rainfall) were not significant (P > 0.05), indicating that these ecological variables were not responsible for the observed genetic differences among populations. The Bangi adult population exhibited a higher level of observed heterozygosity but lower fixation indices in comparison to its seedling population. All other seedling populations also showed positive fixation indices (F), indicating a general excess of homozygotes. This also may suggest selection against homozygotes between the seedling and adult stages. A low level of population differentiation was detected (GST = 0.117 with the Lambir population and GST = 0.085 without the Lambir population). Furthermore, gene flow (Nm) between populations was not significantly correlated with geographical distances for the populations within Peninsular Malaysia. Cluster analysis also did not reflect geographical proximity and gave little insight into the genetic relatedness of the populations. This may indicate that the populations sampled are part of a continuous population with fragmentation having occurred in the recent past.