The crucial role of reproductive isolation in speciation has long been recognized; however, a limited number of studies quantify different isolation barriers and embed reproductive isolation in a phylogenetic context. In this study, we investigate reproductive isolation between the often sympatrically occurring orchid species, Gymnadenia conopsea and G. odoratissima. We examine the phylogenetic relationship between the two species and analyse floral isolation, fruit set and seed viability from interspecies crosses, as well as the ploidy level. Additionally, we quantify interspecies differences in floral signals and morphology. The results suggest that the two species have a sister–species relationship. In terms of reproductive isolation, we found complete floral isolation between the two species, but little to no post-pollination isolation; the species also mostly had the same ploidy level in the studied populations. We also show clear distinctions in floral signals, as well as in floral size and spur length. We propose that respective adaptation to short- vs. long-tongued pollinators was the driver of speciation in the here studied Gymnadenia species. Our study supports the key role of floral isolation in orchid speciation and shows that floral isolation is not restricted to highly specialized pollination systems, but can also occur between species with less specialized pollination.