The morphological variation and cytotype diversity were investigated among Central European populations traditionally recognized as Dactylorhiza fuchsii, recently incorporated in D. maculata s.l. Flow cytometry was employed to assess the ploidy levels of 738 individuals from 77 localities and multivariate morphometrics for a total of 531 individuals from 27 localities. Three ploidy levels were found: diploid (2n = 2x = 40), DNA-triploid and tetraploid (2n = 4x = 80). Whereas diploids and tetraploids often occurred as pure-cytotype populations, individuals of DNA-triploids always co-occurred with at least one of the other cytotypes. Qualitative morphological traits were inferred to be the most important drivers of morphological variation among the investigated plants, with the most striking differences in flower colouration and leaf spotting. The combination of morphological and cytological characters enabled to delimit two separate groups of populations. The first corresponded to D. maculata subsp. fuchsii with morphologically indistinguishable diploid, DNA-triploid and tetraploid individuals, sometimes occurring in mixed-ploidy populations. A complex geographical pattern of cytotype distributions was observed, with diploids scatteredly occurring throughout Central Europe except for Bohemian Massif, which was dominated by tetraploids. The other group of populations represented newly described in this study D. maculata subsp. sooana, subsp. nova, morphologically well-defined and strictly diploid taxon with a restricted geographical range, occurring in the Western Carpathians. A new combination for a hybrid taxon D. × dinglensis nothosubsp. smitakii, comb. nova (= D. maculata subsp. sooana × D. majalis subsp. majalis), was also proposed.