Sequence data from a portion of the external transcribed spacer (ETS) and from the internal transcribed spacers (ITS1 and ITS2) of 18S-26S nuclear ribosomal DNA were used together with chloroplast DNA PCR-RFLP data to unravel patterns of allotetraploid speciation within the Western European Dactylorhiza polyploid complex. A maximum likelihood tree based on combined ETS and ITS sequences suggests that the Western European Dactylorhiza allotetraploids have evolved by hybridization between four main diploid lineages. Cloned sequences and the topology of the ITS plus ETS tree indicate that the allotetraploid species D. elata, D. brennensis, and D. sphagnicola have originated from the autotetraploid D. maculata together with the diploid D. incarnata, while D. majalis, D. traunsteineri, and D. angustata seem to have evolved by hybridization between the D. fuchsii s.str and D. incarnata lineages. Finally, the diploid D. saccifera lineage seems to have been involved together with the D. incarnata lineage in the formation of the allotetraploid D. praetermissa. The observed congruence between the chloroplast tree and the ITS/ETS tree suggests a directional evolution of the nrDNA after polyploidization in favor of the maternal genome. Considered together with morphological, biogeographical, and ecological evidence, the molecular analysis leads us to recognize four species within the investigated allotetraploid complex, namely D. majalis, D. praetermissa, D. elata, and D. sphagnicola.