Abstract Background Little is known about the prevalence of asymptomatic leishmaniasis in Venezuela. The objective of this study was to quantify Leishmania asymptomatic infection in six endemic foci of cutaneous leishmaniasis (CL) in Portuguesa State, Venezuela, where no previous data were available. Methods Study of the prevalence of Leishmania asymptomatic infection was carried out in 841 individuals from six endemic foci of CL in the municipalities Sucre and Ospino, Portuguesa State. We applied the leishmanin skin test (LST) and the internal transcribed spacer 1 (ITS1) PCR to DNA from sera and blood clots of all LST-positive and 20% of LST-negative patients. Results Of 841 inhabitants tested by LST, 197 returned a positive reaction (23.42%); all of the LST-positives (197) and 121 negatives were screened by nested PCR using serum and blood clots. Among the LST-positive group, 2.54% were PCR-positive with sera, while 44.67% were positive with blood clots. In the LST-negative group, PCR was positive in 2.48% of serum samples and in 38.84% of blood clots. Conclusions It is recommended that LST and PCR on blood clots are used together to detect exposure and asymptomatic infection and for identification of the Leishmania species.