BACKGROUND: The screening of Dutch blood donations for West Nile virus (WNV) may be imminent, as WNV emerges in nearby countries and more donors travel to WNV-affected regions. Since 2016 the related, mosquito-borne Usutu virus (USUV) causes seasonal mortality in Dutch birds. To what extent will human USUV infections affect Dutch WNV donor screening?. STUDY DESIGN AND METHODS: From April through September 2018, plasma samples from blood donations in blackbird-rich regions were stored. When increased bird mortality was reported in August, samples from July, August, and September were tested for USUV-RNA in pools of eight, using a home-brew combined WNV/USUV-PCR assay. Reactive pools were deconstructed. Original plasma units and samples of previous and follow-up donations of reactive donors were tested for USUV- and WNV-RNA, and for antibody responses. RESULTS: The number of USUV RNA-positive, WNV RNA-negative donations was 0 of 2688 donations in July, 6 of 4416 in August (1:736), and 1 of 4936 in September. The seven infected donors tested negative for USUV-RNA in preceding and follow-up donations. For 6 donors, seroconversion for USUV-antibodies was demonstrated. All index donations tested positive in a commonly used PCR-assay for WNV donor screening. Three exposed recipients did not show signs of infection. Screening a random subset of 1092 donations from September for USUV-IgG antibodies showed that 22 donors tested reactive; for three donors retrospective testing identified an USUV PCR-positive pre-seroconversion donation. CONCLUSION: Seasonal USUV infection in Dutch blood donors is common. Cross-reactivity in molecular assays for WNV-screening occurs, but can be resolved using USUV- and WNV-specific PCR-primers and sequencing of viral RNA.