Background Mosquitoes play an important role in the transmission of arboviruses including neuroinvasive West Nile virus. (WNV). Despite reports on seroepidemiological evidence and distribution of potential vectors of WNV in Iran, its transmission and dominant lineage(s) in mosquitoes of the Southern region has not been yet investigated. This study was conducted to inventory mosquitoes in Hormozgan province and explore them for WNV infection. Methods A total of 6785 larvae and adult mosquitoes were collected from 11 sites during 2015–2016. The mosquitoes were analyzed for WNV lineage detection by RT-PCR. Results This study revealed vertical transmission of Lineage 1a WNV in five groups including two groups of Cx. pipiens collected in winter and spring as well as three others containing Cx. sitiens, Anopheles stephensi and Cs. longiareolata collected in autumn from Bandar Abbas city. Conclusion The vertical transmission of WNV by effective vectors indicates the incursion of enzootic transmission in the region. The cross-species WNV transmission and prevalence of infected migratory birds coming from Europe and Siberia demand a comprehensive surveillance of WNV infection in hosts and vectors. However, vector-based surveillance remains the most accurate and feasible indicator to assess the impending risk of WNV in Iran. Given the serological and molecular evidences of WNV presence in humans, equines and birds in 26 provinces, the public health authorities should pay more attention to vector control measures to reduce the risk of WNV epidemics in the southern region of Iran.