This nationwide cross-sectional study estimated the seroprevalence of *Borrelia burgdorferi* sensu lato in 716 clinically healthy horses across all 17 autonomous communities of Spain, using ELISA + Western blot confirmation, and identified horse- and management-level risk factors via mixed-effects logistic regression.
Overall crude seroprevalence was 9.6% (69/716), rising to 11.5% after census weighting; seroprevalence was highest in northern regions. Sport horses, horses in northern Spain, and those both tick-exposed and seropositive for *Babesia caballi* had the greatest odds of seropositivity. PCR positivity in whole blood was only 3%, reflecting low sensitivity in that matrix.
PCR on whole blood has limited sensitivity for *Borrelia* detection, likely underestimating active infection. The study sampled only clinically healthy horses, so findings may not reflect diseased populations. Causality cannot be inferred from this cross-sectional design.
Clinicians in northern Spain should maintain a higher index of suspicion for Lyme borreliosis in horses, especially sport horses with tick exposure or concurrent *Babesia caballi* seropositivity. A two-tier serological approach (ELISA then Western blot) is recommended, as PCR on whole blood alone will miss most exposed animals.
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