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Persian leopard (<em>Panthera pardus saxicolor</em>) is the largest <em>P. pardus</em> subspecies in the world. But according to the sharp decline and extinction of populations in some areas, more attention to this subspecies in aspect of genetic conservation is of utmost importance. The goal of this study was to genetic and phylogenetic analysis of D-Loop region in Persian Leopard. Six biological samples of leopard bone and skin were prepared from Tandooreh National Park in Khorasan Razavi province from 2008 to 2011. After DNA extraction, 800bp fragment of the mitochondrial D-Loop was amplified with universal primers, L15926 and H16498 under polymerase chain reaction. Amplified fragments were sequenced with ABI 3130 automated machine under Sanger method. After editing the sequences, the results were compared with sequences from other studies to determine the genetic distances. Variations were not observed in the nucleotide sequences obtained in this study. Using the Neighbor-Joining phylogenetic test results showed that the Persian leopard is in group of <em>P. pardus</em> and its haplotype is the same with <em>saxicolor</em>.