Past climatic changes and especially the ice ages have had a great impact on both the distribution and the genetic composition of plant populations, but whether they promoted speciation is still controversial. The autopolyploid complex Biscutella laevigata is a classical example of polyploidy linked to glaciations and is an interesting model to explore migration and speciation driven by climate changes in a complex alpine landscape. Diploid taxa survived the last glacial maximum in several never-glaciated areas and autotetraploids are clearly dominant in the central parts of the Alps; however, previous range-wide studies failed to identify their diploid ancestor(s). This study highlights the phylogeographical relationships of maternal lineages in the Western Alps and investigates the polyploidy process using plastid DNA sequences (trnS-trnG and trnK-intron) combined with plastid DNA length polymorphism markers, which were transferable among Brassicaceae species. Twenty-one distinct plastid DNA haplotypes were distinguished in 67 populations densely sampled in the Western Alps and main lineages were identified by a median-joining network. The external Alps harboured high levels of genetic diversity, while the Central Alps contained only a subset of haplotypes due to postglacial recolonization. Several haplotypes were restricted to local peripheral refugia and evidence of in situ survival in central nunataks was detected by the presence of highly differentiated haplotypes swamped by frequent ones. As hierarchical genetic structure pointed to an independent evolution of the species in different biogeographical districts, and since tetraploids displayed haplotypes belonging to different lineages restricted to either the northern or the southern parts of the Alpine chain, polytopic autopolyploidy was also apparent in the Western Alps.