Leishmaniasis is one of the major vectors borne parasitic disease recognized by World Health Organization (WHO) and this disease is endemic in tropical and sub-tropical countries of the world including Pakistan. There are three main clinical forms of Leishmaniasis caused by different species of parasite belonging to the genus Leishmania. The detection of Leishmania parasites can be confirmed by various methods like Direct Agglutination Test (DAT), Enzyme Linked Immuno-sorbent Assay (ELISA), Immunofluorescence Assay (IFA), Polymerase Chain Reaction (PCR), direct microscopy and culture technique. In vitro cultivation of parasite plays an important role in study and treatment of the disease. The objective of present study was to evaluate the positivity of direct microscopy and culture technique for detection of Leishmania parasite. The study and laboratory investigations were conducted at Centre of Excellence in Science and Technology (CESAT), Islamabad- Pakistan from January, 2001 to December, 2003. Parasites were isolated from suspected patients to Cutaneous Leishmaniasis (CL) referred by local hospital. Most of these patients were deployed in different regions of Baluchistan where Cutaneous Leishmaniasis is endemic. They were all males & age ranged from 21 - 52 years. They had one or more nodules/ lesions mainly on exposed areas of the body. History of visited in endemic area was one of the inclusion criteria. Organisms were isolated from material obtained by needle aspiration or from biopsy samples from the edges of the lesions using insulin syringe. The biopsy material was homogenized in sterile normal saline. Needle aspirates and tissue homogenates were inoculated into Novy, McNeal Nicole (NNN) Medium. The solid phase of the NNN media contained fresh, aseptically collected defabrinated rabbit blood, mixed with agar and gentamicin. The liquid phase NNN media comprised of 0.85% physiological saline. The Isolates were incubated at 22 - 25°C. Microscopy was done after 48 hours and repeated after 72 hours & than till 02 week for presence of promastigotes. For all patients, the questionnaire was filled. The recorded data included important clinical & social details. Saline aspirate was obtained from 232 patients suspected to CL. Out of 232 samples 87(37.5%) had positive results after microscopic analysis while 138(59.4%) samples showed negative results on microscopic examination and 07(3.017%) cultures were contaminated. These Local isolated Leishmania strains were identified as Leishmania major. The virulence of these organisms improves through animal passage. These Leishmania cultures are being maintained in NNN media for further R & D work on Cutaneous Leishmaniasis.