For some species, particularly invertebrates, developing single copy nuclear markers is an expensive and time-consuming task that may result in few or no usable markers. This has proven true for Onychophora (velvet worms). Here we describe our PCR-based method of generating single copy nuclear markers in Onychophora—a phylum comprised of species generally regarded as rare and of high conservation value—for which suitable microsatellites have been difficult to obtain. We list 6 primer pairs, some of which amplify across genera, and demonstrate their utility in identifying strong population structure in the species Phallocephale tallagandensis.