Ophrys iricolor and O. mesaritica are a pair of morphologically similar, closely related sexually deceptive orchids from the eastern Mediterranean. Ophrys iricolor is known to be pollinated by Andrena morio males and the specific pollinator of Ophrys mesaritica is determined as Andrena nigroaenea. Amplified fragment length polymorphism revealed O. iricolor and O. mesaritica to be genetically intermixed on the whole, although populations of O. iricolor and O. mesaritica in geographical proximity are strongly differentiated, suggesting that specific pollinators locally differentiate these taxa. Based on the available biological data and the system of pollinator attraction operative in Ophrys, we hypothesize that O. mesaritica may have arisen from O. iricolor by pollinator shift and that this is more probable than scenarios invoking hybridization as a result of mispollination by rare, non-specific flower visitors or specifically attracted insects. © 2009 The Linnean Society of London, Botanical Journal of the Linnean Society, 2009, 159, 583–598.