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Background: Asymptomatic infection is the most common outcome of exposure to Leishmania parasites. In the Mediterranean region, where Leishmania infantum is endemic, studies on the prevalence of asymptomatic infection have often relied on serological testing in blood donors. In Spain, regional studies have shown seroprevalence in blood donors between 1 and 8%; in Portugal, values of 0 and 2% were suggested by two localized studies, in different populations. The purpose of this study was (i) to estimate the prevalence of asymptomatic Leishmania infection in blood donors in mainland Portugal, and (ii) to study the association between the detection of antibodies to Leishmania and sociodemographic factors, and also the knowledge, perceptions and practices (KPP) of the blood donors regarding leishmaniasis., Methods: A cross-sectional study targeted the population of people who donated blood in mainland Portugal. Participants, distributed proportionally by municipality and aged between 18 and 65 years, were selected randomly in 347 blood collection points between February and June 2022, and completed a sociodemographic and a KPP questionnaire. Detection of anti-Leishmania antibodies in serum was performed using an ELISA commercial kit. Individual KPP scores were calculated by adding grades defined for each question., Results: Globally, 201/3763 samples were positive. The estimated national true seroprevalence was 4.8% (95% CI 4.1-5.5%). The proportion of positive results was significantly different between NUTS (Nomenclature of Territorial Units for Statistics) regions. Models suggested that seropositivity was significantly higher in male sex, people older than 25 years, or residing in the Centro NUTS2 region, but not in dog owners nor people with lower KPP scores. Overall, 72.3% of participants had previously heard of leishmaniasis and, in multivariate analysis, a higher Knowledge score was associated with age 25-40 years, female sex, ownership of dogs, and higher education., Conclusions: Global estimated true seroprevalence (4.8%) was similar to previous regional studies in blood donors in neighboring Spain. Higher seroprevalence values in the NUTS2 Centro region were consistent with incidence data from humans and seroprevalence studies in dogs. On the other hand, the low values in the Alentejo and the high values in the northern subregions may be the result of geographical shifts in parasite circulation due to climate change and should prompt localized and integrated, vector, canine, and human research, following a One Health approach., (© 2023. BioMed Central Ltd., part of Springer Nature.)