In the present study the genetic variation of the Critically Endangered velvet worm species Opisthopatus roseus is examined. This species is endemic to the Ngele mistbelt forest in the KwaZulu-Natal province of South Africa. In recent years the forest has been severely impacted by anthropogenic activities such as logging of indigenous trees, construction of a national highway, commercial timber plantations and introduction of alien plant species, resulting in habitat fragmentation and potential range contraction for the species. A total of 35 specimens were collected from five sample sites (logs) in the Ngele Forest and sequenced for a partial fragment of the cytochrome c oxidase subunit one (COI) locus. In addition, the sex ratio at each sample sites was determined. A highly skewed sex ratio was detected within sites and only three males were present among the 35 O. roseus specimens. The results reveal the presence of 11 haplotypes and moderate genetic differentiation between the five sampled sites. In addition, it is evident that samples from the same log represented a mixture of haplotypes and did not comprise a single maternal haplotype. The results further suggest that gene flow between individuals occurs in the interior of the forest. Conservation implications of the present study are briefly discussed.