Visceral leishmaniasis is one of the diseases with the greatest impact on public health. In Paraguay, it is transmitted mainly by Lutzomyia longipalpis, which is considered the main vector of Leishmania infantum. In this paper, we report for the first time the detection of Leishmania spp. DNA by real-time PCR in a specimen of Evandromyia (complex) cortelezzii in Alto Paraná-Paraguay. The PCR product was sequenced and showed 100% similarity with Leishmania infantum. Lutzomyialongipalpis is reported for the first time in the Alto Paraná department and 11 out 47 (23,4%) were positive for Leishmania spp. DNA. Besides it was identified for the first time Mycropygomyia quinquefer and Brumptomyia cunhai in this region and without natural infection. In areas where Lutzomyia longipalpis has not been reported or its abundance is very low, other alternative vectors may be involved in parasite transmission. Therefore, although the detection of Leishmania DNA does not incriminate these species as vectors, these results suggest a potential role of these and other phlebotominein the transmission of visceral leishmaniasis in the study area.