Interspecific hybridization and polyploidization have played central roles in plant diversification. However, technical difficulties in the analyses of low-copy genes have limited the study of the origins of hybrid and polyploid plants. Here, we present a phylogenetic analysis of the hexaploid Cardamine asarifolia, distributed in the southern European Alps and northern Apennines. Our study included all relevant taxa of the genus found in Europe. A marked discrepancy was revealed between the trnL-trnF region of cpDNA and internal transcribed spacer (nrDNA ITS) sequences. To solve the incongruence, we sequenced a single-copy nuclear CHS gene (chalcone synthase) using a novel method to design homoeologue-specific PCR primers to bypass artefacts caused by artificial recombination of homoeologues during PCR and/or cloning. Three homoeologues were isolated from C. asarifolia, providing evidence for its allopolyploid origin. One homoeologue, showing the same phylogenetic position as the ITS sequences, most likely originated from an extinct parent. Furthermore, we documented recurrent polytopic hybridizations between C. asarifolia and diploid C. amara. The allohexaploidization and the following hybridization with a diploid species exemplify the ongoing dynamic processes of speciation in the genus Cardamine.