The characteristic of donkey population of Marathwada region is grey to brown coat colour and typical features include white belly, eye-ring, area around the muzzle and inner part of fore and hind limb. In present study, a panel often microsatellite markers viz., AHT5, HTG6, HTG7, HTG10, HMS2, AHT4, NVHEQ54, COR18, COR7 and COR71 were successfully amplified and used for defining genetic variability in the population. The mean allelic richness was 5.44 +/- 1.72, which indicates a considerable amount of variability in the population. The mean Ho and He for the population was 0.694 and 0.802 respectively. Heterozygosity value (H) (0.802) also indicated copious genetic variability and suggest that this genetic resource has a high conservation value. All the ten markers supplemented ample degree of information that is indicative from the Shannon index (I) value of 1.915 with evenness 0.831. The population was found to deviate significantly from HW proportions at all loci (p < 0.0001) except for HMS2. Polymorphic information content (PIC) value for the markers ranged from 0.663 (COR7) to 0.87 (HTG10) suggesting the suitability of these markers for genetic variability studies in the donkey population of Marathwada region. There was absence of any recent genetic bottlenecks in the studied population and a PE value of 1 indicated suitability of these 10 loci for parent exclusion studies in the population. The study suggests that the population of donkey in Marathwada region has a potential to be recognized as a breed.