Podophyllum hexandrum (Indian Mayapple) is an important medicinal plant valued all over the world. Genetic diversity among the 28 genotypes of P. hexandrum distributed in 11 geographical regions from Himachal Pradesh (a part of the North-western Himalaya) was analyzed using RAPD markers. The genetic diversity was high among the genotypes as measured by percentage of polymorphic bands (PPB = 92.37%) and Shanon information index (I = 0.50). The mean coefficient of gene differentiation (Gst) was 0.69, indicating that 33.77% of the genetic diversity resided within the genotypes. Analysis of molecular variance indicated that the source of variation among the groups was 53% and among the genotypes of groups was 47%. An overall value of mean estimated number of gene flow (Nm = 0.22) indicated that there was limited gene flow among the genotypes. The existence of variation among the 28 genotypes as observed through podophyllotoxin content proved to be coupled with geographical altitude (r = 0.92) and local ecological conditions (temperature, rainfall, humidity, soil pH, etc.) but not on genetic basis (r = -0.55). Based on the observed genetic variations among the genotypes of Podophyllum, we recommend for their in situ conservation and germplasm collection expeditions in future conservation plans.