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Objectives: This study was performed to determine the presence of West Nile virus (WNV) in mosquito specimens and human blood donors in Pakistan. Methods: A total of 4150 mosquito specimens were collected using CO2-baited traps from five selected districts of Punjab Province, Pakistan. The mosquitoes were taxonomically identified using standard morphological keys, resulting in 166 pools. In addition, 1070 serum samples were collected from human blood donors. RNA was extracted from mosquito and human samples and screened for WNV using a reverse transcriptase PCR (RT-PCR). Results: None of the mosquito pools tested positive for WNV, whereas three samples from asymptomatic humans tested positive. To determine the WNV strains, partial sequences were compared against a global representation of 23 WNV sequences. The study strains were determined to come from WNV lineage 1. Conclusions: This study is novel in reporting the circulation of lineage 1 WNV in Pakistan. Given its ability to transmit from human to human via blood transfusion, this highlights the urgent need for nationwide surveillance to assess the distribution and impact of WNV in Pakistan. Determining the source of human infection will require more extensive mosquito sampling. Keywords: West Nile virus, Mosquitoes, Human, Pakistan