AbstractEffective protection of endangered species is often limited by taxonomic discrepancies across state borders. This is also the case of the Dactylorhiza maculata agg. in Central Europe, where one to three species and several infraspecific taxa are recognized in various countries. Based on an extensive analysis of morphological variation, ploidy levels, environmental traits and habitats of 64 populations in Central Europe and adjacent regions, we aimed to propose a unified taxonomic concept applicable throughout the study area. Multivariate analysis of morphological traits revealed continuous variation at the individual level and only minor differences between particular clusters of populations. Four DNA-ploidy levels were detected using flow cytometry. Diploids (2n = 40) and tetraploids (2n = 80) were the most abundant and usually formed single-cytotype populations whereas DNA-triploids and DNA-hexaploids occurred only sporadically as minority cytotypes. The inferred patterns of morphological and ploidy variation were not congruent with traditional taxonomic treatment regarding diploid D. fuchsii and tetraploid D. maculata as two species with several infraspecific taxa. Instead, all taxa analysed in the current study are best treated at the subspecies level within D. maculata s. lat. due to somewhat continuous morphological variation between morphotypes. A total of eight D. maculata subspecies may be recognized in Central Europe, of which one is newly described here as D. maculata subsp. arcana, subsp. nov. Some nomenclatural riddles have been resolved, and the threat status of the recognized taxa is discussed.