Chloroplast DNA sequences were obtained from 331 Asplenium ceterach plants representing 143 populations from throughout the range of the complex in Europe, plus outlying sites in North Africa and the near East. We identified nine distinct haplotypes from a 900 bp fragment of trnL-trnF gene. Tetraploid populations were encountered throughout Europe and further afield, whereas diploid populations were scarcer and predominated in the Pannonian-Balkan region. Hexaploids were encountered only in southern Mediterranean populations. Four haplotypes were found among diploid populations of the Pannonian-Balkans indicating that this region formed a northern Pleistocene refugium. A separate polyploid complex centred on Greece, comprises diploid, tetraploid and hexaploid populations with two endemic haplotypes and suggests long-term persistence of populations in the southern Mediterranean. Three chloroplast DNA (cpDNA) haplotypes were common among tetraploids in Spain and Italy, with diversity reducing northwards suggesting expansion from the south after the Pleistocene. Our cpDNA and ploidy data indicate at least six independent origins of polyploids.