Background: CL is an endemic disease in the littoral zones of the Mediterranean area including Libya.Aim of the study: was to detect Leishmania DNA amplified by PCR from smeared cutaneous lesions stored on microscopic slides compared to conventional microscopicy.Methodology: A total of 250 archived microscopic slides with skin smears from cutaneous lesions of clinically suspected cases of CL attending Dermatology Department, Faculty of Medicine, Tripoli University, Tripoli, Libya were microscopically examined and Leishmania DNA was amplified using PCR.Resultes: Using PCR, 20%(50/250) were positive and only14.4% (36/250) were positive by microscopy. Nominating PCR as reference standard, microscopy showed 72% sensitivity and 93.5% NPV with a substantial agreement with ITS-PCR and an overall accuracy of 94.4%. CL was more prevalent among females than males (22.5%/17.7%), residents of rural than urban (22.2%/18.3%) areas, Libyans than non Libyans (20.4%/13.3%), and house wives and students were the most frequently affected (30% and 22%) profesions but without statistical significance. Children were the least affected (9.8%) while middle age (41 to 60 years) was the most affected (27.7%) age group without statistical significance. Conclusion: None of the studied variables showed association for Leishmania infection. ITS-PCR assay as compared to microscopy, offered a sensitive, specific and faster diagnostic method for Giemsa-stained smears, especially with negative parasitologic examination. Which makes it more economically and technically attractive in highly endemic regions and to bring them from research into daily clinical use.