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The Critically Endangered Arabian leopard Panthera pardus nimr faces severe reduction in population size and is on the brink of extinction. This situation is, to a large extent, a result of human activity. The small populations of this subspecies are restricted to a few areas in the Arabian Peninsula, Israel and Jordan. Information required for conservation of this subspecies, including reliable population estimates and the range of individuals, is currently unavailable. To estimate population size and assign gender to individuals in the population in Israel we used molecular markers in leopard DNA extracted from scats collected in intensive surveys throughout the Judean Desert and the Negev Highlands. This non-invasive mode of sampling, combined with the availability of high-resolution markers (microsatellites) and sex-specific DNA-sequences, was successful in identifying both individuals and gender. The results indicated the existence of a male and two females in the Judean Desert, and four males and one female in the Negev Highlands. Although the non-invasive procedure we used may underestimate the leopard’s true population size, continuous monitoring of population size and sex composition of this small population using scatology is a key component for the management of this species. These data, especially if used in conjunction with similar data from other countries within the subspecies’ range, will assist in the establishment of conservation plans for the Arabian leopard.