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Genetic diversity is the basis of the evolutionary potential of species to respond to environmental changes. However, restricting the movement of species can result in populations becoming less connected which can reduce gene flow and can subsequently result in a loss of genetic diversity. Urban expansion can lead to the fragmentation of habitats which affects the ability of species to move freely between areas. In this study, the genetic diversity of the African clawless otter (Aonyx capensis) in Gauteng (South Africa) was assessed using non-invasive sampling techniques. DNA was extracted from spraint (faecal) samples collected along nine rivers and genotyped using 10 microsatellites to assess population structure and genetic diversity. Samples were grouped based on locality and by catchment to determine whether isolated subpopulations exist. Genetic diversity of A. capensis in Gauteng was found to be low (mean observed heterozygosity (Ho)=0.309). Analysis of genetic structure provides support for the otter populations being panmictic with high gene flow between populations from different rivers. Results from the study indicate that the movement of A. capensis is not affected by physical barriers in urbanised areas. However, because the genetic diversity of the species in the study area is low, these animals may not be able to cope with future environmental changes.