Background Recent studies highlighted the role of Pleistocene climatic cycles in polyploid speciation and of southern Alpine refugia as reservoirs of diversity during glacial maxima. The polyploid Primula marginata, endemic to the southwestern Alps, includes both hexaploid and dodecaploid cytotypes that show no ecological or morphological differences. We used flow cytometry to determine variation and geographic distribution of cytotypes within and between populations and analyses of chloroplast (cp) and nuclear ribosomal (nr) DNA sequences from the Internal Transcribed Spacer (ITS) region to infer the evolutionary history of the two cytotypes and the auto- vs. allopolyploid origin of dodecaploid populations. Results We did not detect any intermediate cytotypes or variation of ploidy levels within populations. Hexaploids occur in the western and dodecaploids in the eastern part of the distributional range, respectively. The cpDNA and nrDNA topologies are in conflict, for the former supports shared ancestry between P. marginata and P. latifolia, while the latter implies common origins between at least some ITS clones of P. marginata and P. allionii. Conclusions Our results suggest an initial episode of chloroplast capture involving ancestral lineages of P. latifolia and P. marginata, followed by polyploidization between P. marginata-like and P. allionii-like lineages in a southern refugium of the Maritime Alps. The higher proportion of ITS polymorphisms in dodecaploid than in hexaploid accessions of P. marginata and higher total nucleotide diversity of ITS clones in dodecaploid vs. hexaploid individuals sequences are congruent with the allopolyploid hypothesis of dodecaploid origin.