Borrelia burgdorferi was found widespread in ixodid ticks from the Basque Country (Spain) during a two-step study. In the first part, a total of 7,835 ixodids of eight different species was collected from vegetation, classified, and processed using polymerase chain reaction (PCR) for detection of B. burgdorferi ospA DNA. B. burgdorferi DNA was detected in ≤12.5% of adults and ≥0.6% of Ixodes ricinus (L., 1758) nymphs (mean 1.5 and 0.05%, respectively), and in ≤14.3% of adult Hemaphysalis punctata (Canestrini & Fanzago, 1877) analyzed (mean 1.2%). The second part of the study was undertaken 2 yr later to characterize B. burgdorferi distribution by focusing on the areas where I. ricinus was the predominant species. Ten areas were selected from which 1,535 nymphs and adults of I. ricinus were collected and processed by PCR and culture techniques. Infected ticks were found in all zones. B. burgdorferi DNA was detected in a mean of 9.3 and 1.5% of adults and nymphs, respectively. Nine isolates of B. burgdorferi were obtained, belonging to four different genospecies (B. burgdorferi sensu stricto, B. garinii, B. valaisiana, and B. lusitaniae). The results indicate that some areas of Spain have a potential risk for Lyme disease agent exposure and that B. burgdorferi appears to have an increasing occurrence in ticks in the Basque Country.