Usutu virus (USUV) is a flavivirus (Flaviviridae: Flavivirus) of an African origin transmitted among its natural hosts (diverse species of birds) by mosquitoes. The virus was introduced multiple times to Europe where it caused mortality of blackbirds (Turdus merula) and certain other susceptible species of birds. In this study, we report detection of USUV RNA in blackbirds, Culex pipiens and Cx. modestus mosquitoes in the Czech Republic, and isolation of 10 new Czech USUV strains from carcasses of blackbirds in cell culture. Multiple lineages (Europe 1, 2 and Africa 3) of USUV were found in blackbirds and mosquitoes in the southeastern part of the country. A single USUV lineage (Europe 3) was found in Prague and was likely associated with increased mortalities in the local blackbird population seen in this area in 2018. USUV genomic RNA (lineage Europe 2) was detected in a pool of Cx. pipiens mosquitoes from South Bohemia (southern part of the country), where no major mortality of birds has been reported so far, and no flavivirus RNA has been found in randomly sampled cadavers of blackbirds. The obtained data contributes to our knowledge about USUV genetic variability, distribution and spread in Central Europe.