Accurate diagnosis of cutaneous leishmaniasis (CL) is important for chemotherapy and epidemiological studies. Common approaches for Leishmania detection involve the invasive collection of specimens for direct identification of amastigotes by microscopy and the culturing of promastigotes from infected tissues. Although these techniques are highly specific, they require highly skilled health workers and have the inherent risks of all invasive procedures, such as pain and risk of bacterial and fungal super-infection. Therefore, it is essential to reduce discomfort, potential infection and scarring caused by invasive diagnostic approaches especially for children. In this report, we present a novel non-invasive method, that is painless, rapid and user-friendly, using sequential tape strips for sampling and isolation of DNA from the surface of active and healed skin lesions of CL patients. A total of 119 patients suspected of suffering from cutaneous leishmaniasis with different clinical manifestations were recruited and samples were collected both from their lesions and from uninfected areas. In addition, 15 fungal-infected lesions and 54 areas of healthy skin were examined. The duration of sampling is short (less than one minute) and species identification by PCR is highly specific and sensitive. The sequential tape stripping sampling method is a sensitive, non-invasive and cost-effective alternative to traditional diagnostic assays and it is suitable for field studies as well as for use in health care centers.