The Leptynia hispanica stick insect species complex includes bisexuals, triploid and tetraploid parthenogenetic populations, suggesting that polyploidy has played a central role in the evolution of this complex. An analysis of karyotype, mitochondrial DNA (cox2) and nuclear DNA (ef1-α) markers was carried out to clarify phylogenetic relationships and microevolutionary/phylogeographical patterns of the L. hispanica complex. Our analyses suggested a subdivision of bisexual populations into four groups, tentatively proposed as incipient species. Moreover, triploids and tetraploids showed two independent origins, the latter being more ancient than the former. From ef1-α analysis, triploids showed hybrid constitution, while the hybrid constitution of tetraploids is likely, but more data are needed. We suggest that L. hispanica is a case of ‘geographical parthenogenesis’ with parthenogenetic strains colonizing large peripheral ranges, and bisexuals confined to glacial refuge areas. Moreover, the age, wide distribution and competitive advantage of polyploids over diploids, demonstrate their significance in the evolution of the L. hispanica species complex.