The pattern of genetic variation among populations of two Orobanche gracilis Sm. taxa (var. gracilis and var. deludens (Beck) A. Pujadas) from Northern and Southern Spain growing on different hosts was analysed using RAPD markers. The diversity analysis within populations revealed a higher level of diversity in the populations from the North when compared to the Southern ones. The results of principal co-ordinate analysis (PCoA) based on Dice distances among samples clearly established the separation of samples according to the taxonomical variety and the geographical origin of each population. The Southern populations of both var. gracilis and var. deludens were more differentiated among them than those of var. gracilis from the North. The analysis of molecular variance (AMOVA) indicated that the lowest level of population differentiation was found in O. gracilis var. gracilis from the North, whereas in the case of O. gracilis var. deludens from the South most of the genetic diversity was attributable to differences among populations. Possible explanations for the distribution of variation in these populations are discussed.