The prevalence of Leishmania infantum-specific antibodies and asymptomatic infection was assessed in a randomized sample of 526 healthy adults from a continental area of northwestern Italy where L. infantum is not endemic and where autochthonous cases of visceral leishmaniasis (VL) were recently reported. L. infantum-specific antibodies were detected by Western blotting (WB) in 39 subjects (7.41%), while L. infantum kinetoplast DNA was amplified from buffy coat in 21 out of 39 WB-positive subjects, confirming asymptomatic infection in 53.8% of seropositives. Risk factors significantly associated with WB positivity were uninterrupted residence since childhood in a local rural environment (odds ratio [OR], 3.5; 95% confidence interval [CI], 1.7 to 7.3), daily contact with animals though not exclusively with dogs (OR, 3.7; 95% CI, 1.3 to 10.7), older age (OR, 2.31; 95% CI, 1.2 to 4.5), and agricultural/other outdoor activities (OR, 3.8; 95% CI, 0.99 to 3.7.) Logistic regression analysis showed that uninterrupted residence in a local rural environment and an age of >65 years were the only independent predictors of seropositivity assessed by WB. Follow-up at 24 months did not show evidence of VL in either seropositive or PCR-positive subjects. The detection of a high seroprevalence rate, confirmed as asymptomatic infection by PCR in more than half of the cases, among healthy residents in a continental area of northwestern Italy makes local L. infantum transmission very likely. In a region where VL is considered nonendemic, these findings warrant further epidemiological investigations as well as interventions with respect to both the canine reservoir and vectors, given the possible risks for immunosuppressed patients.