Thirty-four small mammals collected in the vicinity of Ljubljana were tested for the presence of Borrelia burgdorferi sensu lato by polymerase chain reaction (PCR) of urinary bladder tissues, using universal flagellin primers and species specific rRNA primers. Seventeen small mammals (50%) were found to be positive, and 7 small mammals were infected with two species of B. burgdorferi sensu lato simultaneously. The most commonly found species was B. afzelii (n = 14), followed by B. burgdorferi sensu stricto (n = 7) and B. garinii (n = 3), as determined by species-specific primers. We conclude that PCR is a rapid and reliable method to detect infection with B. burgdorferi sensu lato in small mammals.