The objective of the study was to determine the prevalence rate of 3 Borrelia burgdorferi genospecies in Ixodes ricinus ticks collected from wooded areas of the Lublin region (eastern Poland). A group of 1,813 I. ricinus ticks from 6 districts were examined for the presence of Borrelia burgdorferi sensu lato (B.b.s.l.) by polymerase chain reaction (PCR). Another group of 438 I. ricinus ticks collected from 4 districts were examined for the presence of B.b.s.l. by culture on BSKH liquid medium confirmed by PCR, and for the presence of Borrelia spp. by dark field microscopy (DFM). Borrelia burgdorferi genospecies (Borrelia burgdorferi sensu stricto, Borrelia afzelii and Borrelia garinii) were determined by nested-PCR in 113 ticks lysates showing presence of B.b.s.l. (in PCR or in culture and PCR). 5.4 % of I. ricinus ticks examined by PCR showed the presence of B.b.s.l. DNA. The infection rate was highest in females (12.1 %), lower in males (6.0 %) and the lowest in nymphs (1.7 %) (p < 0.001). The minimum infection rate of I. ricinus ticks with B.b.s.l. determined by culture was 3.4 %, whereas the minimum infection rate of ticks with motile spirochetes morphologically resembling Borrelia spp., determined by DFM, amounted to 11.2 %. The presence of all 3 Borrelia burgdorferi genospecies under investigation was found in ticks collected from 5 out of 6 examined districts. In 81.4 % of infected ticks only single infection with 1 genospecies was observed, while coinfections with 2 or 3 genospecies were detected respectively in 16.8 % and 1.8 % of infected ticks. Borrelia burgdorferi sensu stricto was the dominant genospecies in all examined tick stages and districts, both in single infections and in coinfections, and found in a total of 62.8 % of I. ricinus ticks infected with B.b.s.l. Borrelia afzelii and Borrelia garinii were less frequent and observed in respectively 39.8 % and 17.8 % of infected ticks.