This study aimed at finding out the most effective clinical samples and methods in chronic cutaneous leishmaniasis (CCL). Smear, aspiration fluid, and filter paper samples were taken from 104 skin lesions of suspected cases with CCL, and they were compared using microscopic examination, culture, and molecular methods. We characterized four different forms of CCL and identified the causative agents in CCL forms using high-resolution melting curve real-time polymerase chain reaction assay. We observed that smear was detected to be the most sensitive (63.5%) among clinical samples, and real-time polymerase chain reaction method was the most sensitive (96.8%) among the methods used in diagnosis of CCL. We identified 68.8% Leishmania tropica and 31.2% L. infantum in papular lesions, 69.2% L. infantum and 30.8% L. tropica in nodular lesions, 57.9% L. tropica and 42.1% L. major in ulcerating plaque lesions, and 55.5% L. tropica and 44.5% L. major in noduloulcerative lesions in CCL patients.