Seventy-four nucleotide sequences from the ITS regions of nuclear ribosomal DNA and 76 from the trnL-trnF spacer of chloroplast DNA were used to address the origin of tetraploid Cardamine amporitana, the conspecifity of central Italian and northeastern Spanish populations, and the possible cause for such geographic disjunction. Because of the complex lineage relationships in Cardamine, the sampling included 22 taxa. In the results, both data sets are highly congruent in supporting a close relationship of C. amporitana to the widespread Eurasian C. amara. Low genetic variability in northeastern Spanish populations of C. amporitana suggests long-distance dispersal from central Italy. The interior position of the single northeastern Spanish haplotype in a statistical parsimony network of trnL-trnF haplotypes however does not support this scenario and invokes other plausible phylogeographic explanations. The disappearance of geographically intermediate populations and genetic impoverishment by migration and isolation, both probably associated with Quaternary climatic oscillations, appears as an alternative hypothesis to explain the phylogeographic pattern. A recent hybridization event is reported between C. amporitana and a diploid from the C. pratensis group in central Italy on the basis of additive polymorphisms in ITS for all the 22 distinguishing nucleotides.