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We aimed to estimate the seroprevalence of Puumala orthohantavirus (PUUV) among forestry workers in northern France, and to explore sociodemographic risk factors. We conducted a random cross-sectional seroprevalence survey among 1777 forestry workers in 2019–2020. The presence of immunoglobulin G against PUUV antigens in serum was assessed using enzyme-linked immunosorbent assay and confirmed using immunofluorescence assay. Poisson regression models were used to explore factors associated with seropositivity. Weighted seroprevalence was 5% (3–6) in northeastern France, 4% (2–6) in north central France, and 1% in two regions located in the center of the country (Auvergne and Limousin). There were no seropositive workers detected in northwestern France. Seropositivity was associated with age, sex, and cumulative seniority in the forestry sector. Seroprevalence was highest in known endemic areas of the northeast and lowest in the northwest. Nevertheless, we found serological evidence of PUUV infection in two regions located in the center of the country, suggesting circulation of the virus in these regions, previously thought to be non-endemic. [ABSTRACT FROM AUTHOR]