Reproductive barriers play an important role in the maintenance of species boundaries. However, to date few studies have provided a detailed analysis of reproductive isolation barriers between species or examined their importance in maintaining species identity. Recent anthropogenic encroachment has diminished ecological isolation favouring situations of contacts between related species, allowing research into reproductive barriers in natural populations. This is the first detailed study into pre-and postzygotic reproductive isolation barriers in the Antirrhinum genus, based on a mixed population with two species. Antirrhinum valentinum and Antirrhinum controversum are practically completely isolated by both pre-and postzygotic barriers. Prezygotic isolation is slightly greater than postzygotic isolation. Breaking the geographical isolation barrier has less impact on total reproductive isolation and both species are effectively isolated specifically by the action of pollinator constancy and preference and poor seed viability.