Evolutionary relationships in the velvet worm species, Peripatopsis overbergiensis, were examined in 3 forest areas in the Overberg region of South Africa to explore the impact of historical habitat fragmentation on the population genetic structure of the species. We collected 84 P. overbergiensis specimens from Grootvadersbosch, Koppie Alleen, and Marloth Nature Reserves and sequenced all these specimens for the cytochrome c oxidase subunit I (COI) locus, whereas a subset of 13 specimens were also sequenced for the 18S rRNA locus. Phylogenetic analyses of the 20 unique COI haplotypes revealed 4 genetically distinct clades, a result that is corroborated by the haplotype network. A hierarchical analysis of genetic variation was performed on the COI haplotype data within the 2 large forested areas, Grootvadersbosch and Marloth Nature Reserves, and across all 3 of the sample localities. These results revealed low haplotypic and nucleotide diversity within the largest Grootvadersbosch Nature Reserve forest and high haplotypic and nucleotide diversity within the fragmented Marloth Nature Reserve forest, whereas Koppie Alleen had the lowest haplotypic and nucleotide diversity. Across all 3 main localities statistically significant F ST values were found, together with the absence of shared haplotypes indicating the absence of maternal gene flow. Divergence time estimations based on the 20 COI haplotypes calculated in BEAST suggest a Pleistocene/Holocene divergence between the 4 clades as a result of habitat fragmentation and the aridification of the region. Our results indicate that conservation efforts should also prioritize linked, smaller fragmented habitats together with continuous habitats to maximize the genetic diversity of saproxylic fauna.