Lyme borreliosis is an endemic foci disease which occurs in humans and animals and it is caused by various species of pathogenic bacteria within B. burgdorferi s.l. complex. The epizootiological cycle of Lyme borreliosis in nature involves horizontal transmission between vectors and reservoirs. In Europe, the major vector is tick Ixodes ricinus, while small rodents are known as the major reservoirs of B. burgdorferi. The aim of this study was to determine the frequency of infection in small rodents in several endemic areas of Lyme borreliosis in the Republic of Croatia and to determine the possible association of certain rodent species with the particular bacterial species within the Borrelia burgdorferi s.l. complex. Polymerase chain reaction was used to test 197 urinary bladder samples of small rodents. All the samples tested negative for the presence of B. burgdorferi when using touchdown PCR method and primer pairs targeting the large part of flaB gene. We randomly selected 20/197 (10%) urinary bladder samples and tested them again by using touchdown PCR method and primer pairs targeting the small part of flaB gene and also by using nested PCR method and primer pairs targeting the ospA gene. Two (10%) samples of urinary bladder tested positive for the presence of B. burgdorferi when using both protocols. Additionally, other organ samples from both positive rodents were tested and only the samples of ear tissues tested positive for borrelial DNA. Given the number of reported human cases in Croatia and the fact that Lyme borreliosis is the second most common zoonosis in Croatia, further studies on B. burgdorferi reservoirs and vectors are needed to determine the real prevalence of bacterial species within B. burgdorferi s.l. complex in these animals that are essential for maintaining the enzootic cycle of Lyme borreliosis in the Republic of Croatia.