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Not AvailableThis study conducted with a panel of 24 polymorphic microsatellites revealed high number of alleles and heterozygosity in all the Indian donkey clusters available in different agro-climatic regions. All the markers are highly polymorphic as reflected from high allele number and heterozygosity, thus suitable for genotyping studies. Genetic diversity within each donkey population as well as between populations of different regions was also quite high indicating no extinction threat to population. The genetic differentiation suggested that 89.59% genetic variation corresponds to difference among individuals and 10.41% is attributed to differences among population clusters. However conservation and preservations of donkeys is important as their overall population is decreasing rapidly in country. Even the 24 microsatellites utilized for individual assignment did not reveal 100% assignment of donkeys to their clustered population. Breed relationship analysis revealed closeness among Leh and Bihar donkeys which seems to be unique as geographically these populations are at distant places and mixing of these donkeys cluster is not feasible. Closeness of donkey population from Haryana, Rajasthan, Gujarat and Baramati regions may be due to sharing of common breeding tract and movement of donkeys in these agro-climatic regions with their owners during harsh and odd environmental conditions for their survival. Population structure analysis revealed that donkey population from seven regions can be divided in two main clusters: first cluster having donkey population from Bihar and leh while second cluster included donkeys from Gujarat, Rajasthan, Spiti. Population from Baramati and Haryana had more than 50% individual population as admixed while Spiti donkeys had no admixed individuals. The current study aimed to provide insight into the genetic relationships and diversity between various indigenous donkey populations will offer a valuable reference for rational strategies in breed assignment to local non-descript donkeys, donkey conservation and breeding programsNot Availabl