Habitat preference and pollination syndrome have been suggested as major factors in reproductive isolation among plant species. The columbine genus Aquilegia contains species that have been used as classic examples of reproductive isolation due to ecological and floral factors. In this analysis Aquilegia formosa, Aquilegia pubescens, and natural hybrid populations between these two species were assayed for genetic and morphological variation. Clinal variation was evident for three "random amplified polymorphic DNA" loci and five morphological characters along a transect extending from a lower altitude A. formosa population, through an intermediate hybrid population, to a higher altitude A. pubescens population. Similar clinal variation was also discovered for a transect that included A. formosa-like, hybrid, and A. pubescens-like populations at a single elevation. The change in the frequency of both sets of markers was closely associated with change in habitat. The molecular markers indicate the presence of bidirectional introgression between these two species. In contrast, there was apparently selection against introgression of four of the five alternate floral characters. Selection against the incorporation of floral characters from one species into the other species was suggested by the introgression of the DNA markers with little or no introgression of the four floral characters. These findings suggest the importance of adaptations associated with both pollination syndromes and habitat preference on species integrity.