Big cats (Genus: Panthera) are among the most threatened mammal groups of the world, owing to hunting, habitat loss, and illegal transnational trade. Conservation genetic studies and effective curbs on poaching are important for the conservation of these charismatic apex predators. A limited number of microsatellite markers exists for Panthera species and researchers often cross-amplify domestic cat microsatellites to study these species. We conducted data mining of seven Panthera genome sequences to discover microsatellites for conservation genetic studies of four threatened big cat species. A total of 32 polymorphic microsatellite loci were identified in silico and tested with 152 big cats, and were found polymorphic in most of the tested species. We propose a set of 12 novel microsatellite markers for use in conservation genetics and wildlife forensic investigations of big cat species. Cumulatively, these markers have a high discriminatory power of one in a million for unrelated individuals and one in a thousand for siblings. Similar PCR conditions of these markers increase the prospects of achieving efficient multiplex PCR assays. This study is a pioneering attempt to synthesise genome wide microsatellite markers for big cats.