Leishmaniasis is a widespread parasitic disease that occurs as a result of infection with a unicellular parasite belonging to the genus Leishmania. Diagnosis by conventional methods is inaccurate and is not sensitive to confirm the genus infection. Here, we have investigated a methods for Leishmania genus diagnosis, which includes the technique of polymerase chain reaction to detect the presence of the parasite at in vitro for promastigote cultures using three genus-specific primer pairs to amplify HSP70, ITS, and ITS2. The results showed single band of~ 1422,~ 1020, and~ 550 respectively. This study has proved the ability of these primer pairs to detect Leishmania infection and recommend them to be used for detection of leishmaniasis in hospitals and research centers.