Summary The genetic concerns in conservation biology arise from the deleterious effects of small population size, which is a consequence of loss of genetic diversity through genetic drift and inbreeding. The endangered Ethiopian wolf (Canis simensis) inhabits the Ethiopian highlands, with a total species population of about 500 individuals. The total population is sub-divided into the North and the South side of the Great Rift Valley, without any current gene-flow between sub-populations. The northern population is further divided into five isolated sub-populations, with population sizes of less than 50; hence they are at high risk of extinction. In this study, I set out to investigate the levels of microsatellite variation within two of the northern sub-populations, and between the two sides of the Great Rift Valley. Furthermore, I wanted to evaluate the potential of faeces as DNA source for obtaining genetic data from these areas. Based on population genetic theory, my expectations were that these populations would be depleted in genetic variation, and that there would be a strong effect of geographic distance on genetic structure between northern and southern Ethiopia. The amplification success of the faecal samples was low, with 11.7% of the samples giving DNA of sufficient quality for genotyping over 8 microsatellite loci. The main factors affecting amplification success was sample age, humidity and the presence of PCR inhibitors. Within northern Ethiopia, allelic diversity was low, with an allele frequency distribution indicating a recent genetic bottleneck. A small effect of geographic distance on genetic diversity was observed within the North, reflecting the recent isolation of these populations. A prominent effect of population sub-division was observed between northern and southern Ethiopia, with a FST of 0.269. All individuals were assigned to their correct geographic population, with the exception of two individual samples from the North. Sequencing of the mitochondrial Cytochrome b gene revealed that they were different from all other canid sequences published in Genebank. A phylogenetic analysis grouped the unmatched sequences with the group of grey wolf sub-species, which may suggest that they originate from a grey wolf relict. The results of this study illuminate the urgent need for more data from these areas, as further investigation will be crucial for future management of the Ethiopian wolf and for resolving the origin of the peculiar individuals.