| Summary: | In the period from 2015 to 2020, an entomological survey for the presence of West Nile virus (WNV) and Usutu virus (USUV) in mosquitoes was performed in northwestern Croatia. A total of 20,363 mosquitoes were sampled in the City of Zagreb and Međimurje county, grouped in 899 pools and tested by real-time RT-PCR for WNV and USUV RNA. All pools were negative for WNV while one pool each from 2016 (<i>Aedes albopictus</i>), 2017 (<i>Culex pipiens</i> complex), 2018 (<i>Cx. pipiens</i> complex), and 2019 (<i>Cx. pipiens</i> complex), respectively, was positive for USUV. The 2018 and 2019 positive pools shared 99.31% nucleotide homology within the USUV NS5 gene and both clustered within USUV Europe 2 lineage. The next-generation sequencing of one mosquito pool (<i>Cx. pipiens</i> complex) collected in 2018 in Zagreb confirmed the presence of USUV and revealed several dsDNA and ssRNA viruses of insect, bacterial and mammalian origin.
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