Urban areas present a challenging and rapidly changing environment to wildlife, and threats to wild animals include physical harm and persecution, pollution, habitat loss and degradation.  Expanding cities also lead to the fragmentation of habitats and the ability to move freely between areas is greatly reduced if not prevented entirely and leads to deviation from natural movement patterns. In this study the genetic diversity of the Cape clawless otter (Aonyx capensis) in the greater Johannesburg area of South Africa was assessed using non-invasive sampling techniques. DNA was extracted from spraint samples collected along 10 rivers and genotyped using 10 microsatellites per sample to determine relatedness of individuals. Individuals were then grouped based on relatedness and locality to determine whether isolated subpopulations existed. Preliminary results showed that at least two isolated groups occurred, separated by the large city of Johannesburg with subpopulations occurring to the north and south of the city and no obvious conspecific interaction occurring. This study demonstrates the effects of physical barriers such as highways, buildings and fences on the genetic diversity and possibly the movement and reproductive strategies used by this otter species.