We investigated the presence of Borrelia burgdorferi sensu lato in Ixodes ricinus ticks collected in a Lyme borreliosis (LB) endemic region of northern Croatia. Ticks (n = 124) were collected at five locations and analysed by the polymerase chain reaction (PCR). A DNA fragment from the internal transcribed spacer (ITS2) of I. ricinus was detected in all tick lysates, indicating that PCR inhibitors were not present. Borrelia burgdorferi sensu lato DNA was detected in 56 out of 124 ticks (45%). Four genomic groups were identified: Borrelia afzelii (n = 26), Borrelia garinii (n = 5), group VS116 (n = 5) and B. burgdorferi sensu stricto (n = 1). Mixed infections of B. afzelii with group VS116 (n = 10) and B. afzelii with B. burgdorferi sensu stricto (n = 1) were also detected. Eight ticks contained B. burgdorferi sensu lato, which could not be typed. The detection of B. afzelii and B. garinii in ticks was in agreement with manifestations of LB found locally. The occurrence of group VS116 in northern Croatia and in an earlier study in The Netherlands, infers that this genomic group may be well established in European I. ricinus.