Scalesia divisa and S. incisa (Asteraceae), both endemic to the Galápagos Islands, are found only on San Cristóbal, where S. divisa grows in the north-west of the island while S. incisa occurs in the north-east. At localities in between, populations with deviating individuals occur. Here we analyse the population structure of S. divisa, S. incisa and two deviating populations based on morphology, AFLP markers and two plastid microsatellite loci. The deviating populations were collected from either side of the island. In a principal components analysis based on morphological characters they appeared to be intermediate between the presumed pure species. When using a discriminant analysis, the two populations that were geographically furthest apart were best discriminated. A Mantel test showed that there was a significant correlation between morphological differentiation and geographical distance, which was also indicated in a distance tree. A second distance tree based on AFLP characters revealed the same topology, but the branches were longer. This was explained by high within-population variation, as demonstrated by AMOVA. Although only a small proportion of the total variance was explained by the between-population component, the populations were distinct enough to be separated by a discriminant analysis. A high level of misclassification was only found between one of the S. incisa populations and one of the deviating populations. The plastid markers supported the results obtained from AFLP. We hypothesize that the pattern of variation is the result of hybridization between two formerly isolated species.