Populations of endangered mammals are often small, fragmented and have low genetic variability that can reduce the ability to evolve in response to environmental changes. The endangered Ethiopian wolf (Canis simensis) is a habitat specialist restricted to six small, isolated pockets of Afroalpine habitat, with a total population of fewer than 500 adult animals. The degradation of the Ethiopian highlands because of human expansion is ever increasing, potentially leading to further population fragmentation and local extinctions. In order to assist Ethiopian wolf conservation management, we quantified the genetic diversity, population structure and patterns of gene flow of the species using up to 14 microsatellite loci. FST, analysis of molecular variance, principal coordinates analysis and Bayesian clustering analyses revealed geographic population structuring delimited by three mountain ranges, in concert with a previous study based on mitochondrial DNA. Bayesian analysis showed that current gene flow is low, unidirectional and limited to geographically proximate populations. Given the small census size and strong population structuring with low gene flow, demographic stochasticity is likely to be the highest threat to the long-term persistence of this species. The protection of the remaining suitable habitat, especially narrow ridges linking habitat patches within mountain blocks, is therefore essential. The genetic survey presented by this study provides vital and much needed information for the future effective management of Ethiopian wolf populations.