We investigated variation in the Melampyrum sylvaticum group in the Carpathian and Hercynian regions using morphological and molecular tools. The aim of our study was to examine differences in the pattern of variation between the Eastern Carpathians and region of theWestern Carpathians and the Hercynian Massif. We also tested correlations between putatively taxonomically important variation in corolla colour present in the Melampyrum sylvaticum group in the Eastern Carpathian region and other morphological and molecular traits. Samples were collected from populations of the M. sylvaticum group in the Hercynian Massif and the Eastern and Western Carpathians. Morphometric analyses of the size and shape of the corolla (based on thin plate spline with sliding semilandmarks), length of the anthers and especially molecular analyses based on sequencing the nuclear ITS and trnL-trnT regions of chloroplast DNA, confirmed that the populations occurring on the opposite sides of the Eastern-Western Carpathian biogeographic boundary are very different. It is likely that the eastern and western lineages have been isolated for a long time and the extant pattern of variation with character disagreement within the border zone, originated from hybridization and introgression. The differences in corolla colour did not coincide with the variation in morphological traits or molecular markers within the North-Eastern Carpathian region. In addition, the geographical distribution of the populations with contrasting corolla colours lacked any pattern and there are populations with both corolla colours as well as plants with transitional pale-yellow flowers. Therefore, it is suggested that M. saxosum and M. herbichii, microspecies delimited on the basis of corolla colour, are conspecific. The high level of molecular variation and its pattern indicate that the M. sylvaticum group may have survived in or near the Eastern Carpathians during the Weichselian Ice Age. This hypothesis is supported by several recent phytogeographical and palaeoecological studies, which indicate the existence of a glacial refuge in the Eastern Carpathian region. Molecular uniformity of theWestern Carpathian and Hercynian populations might in contrast indicate recent (Holocene) migration from assumed perialpine refuges.