The origin of the obligate-parthenogenetic New Zealand stick insect genus Acanthoxyla was investigated using cytogenetics and sequencing of nuclear and mitochondrial DNA. Little mitochondrial DNA sequence variation (COI-II) was found among seven species of the genus Acanthoxyla and we found no evidence for monophyly of the morphologically distinguished lineages. In contrast, two distinct clades of nuclear sequence (ITS) were obtained, one is restricted to the genus Acanthoxyla, while the other includes sequences obtained from its sister genus Clitarchus. Although Acanthoxyla appears to be diploid (2n = 36-38), it has two ill-matched chromosome pairs. We hypothesize that two or more hybridization events involving the parental sexual species Clitarchus hookeri and an unknown taxon probably resulted in the formation of the parthenogenetic genus Acanthoxyla. However, the karyotype of Acanthoxyla bears little resemblance to the karyotype of the putative paternal species C. hookeri so the exact nature of Acanthoxyla remains in question.