Leishmaniosis, caused by phlebotomine sand fly-borne Leishmania spp., is a multi-host and zoonotic disease in tropical, subtropical and temperate climates, worldwide. Leishmania infantum is the only endemic pathogenic species in Europe, and exposure to this protozoan has previously been reported in a wide range of wild mammals, including felids. However, the information about the exposure of the threatened Iberian lynx (Lynx pardinus) to L. infantum is still very scarce. Consequently, the aims of the present large-scale epidemiological study were: (1) to evaluate the circulation of L. infantum in the free-ranging and captive Iberian lynx populations and (2) to investigate potential risk factors associated with L. infantum exposure during the study period, between 2010 and 2022. A total of 783 Iberian lynxes from captive (n = 296) and free-ranging (n = 487) populations were sampled from different areas of the Iberian Peninsula. Exposure to L. infantum (positive to indirect fluorescent antibody test (IFAT) in serum and/or real-time quantitative PCR (qPCR) in spleen samples) was detected in 80 of the 783 lynxes, so the estimated prevalence (95% confidence limits) was 10.2% (8.1-12.3%). Specifically, anti-Leishmania antibodies were found in 12 of the 469 lynxes (2.6%; 1.1-4.0%) and L. infantum kDNA was detected in 68 of the 333 lynxes (20.4%; 16.1-24.8%) with a Ct range from 26.0 to 38.0 (median: 36.0). One seroconversion and one seroreversion were observed among the 42 Iberian lynxes longitudinally sampled. Phylogenetic analysis revealed high homology (99.9-100%) with other L. infantum sequences obtained from wild rabbits (Oryctolagus cuniculus), Iberian hares (Lepus granatensis) and humans from Spain. The multivariate analysis identified the habitat status (free-ranging) and age (adult and subadult) as risk factors potentially associated with L. infantum exposure in Iberian lynxes. Our results indicate moderate and widespread circulation of this pathogen in the Iberian lynx populations, suggesting its potential role as spillover hosts in Iberian Mediterranean ecosystems. Additional studies are warranted to determine the clinical impact of L. infantum exposure in this threatened species.