From April to October 2007, host-seeking Ixodes ricinus ticks were collected from 4 locations in southern Norway: Farsund, Mandal, Søgne and Tromøy. Two hundred and ten larvae, 1130 nymphs and 449 adults were investigated for infection with Borrelia burgdorferi sensu lato (s.l.) by real-time polymerase chain reaction (PCR) amplification of the 16S rRNA gene. The total percentage of B. burgdorferi s.l. in nymphal and adult ticks was determined to be 31.3% in Farsund, 25.2% in Mandal, 22.3% in Søgne and 22.1% in Tromøy. Larvae were pooled in groups of 10 before analysis, and Borrelia infection was detected in 1 of the 21 larvae pools. B. burgdorferi s.l. were genotyped by melting curve analysis after real-time PCR amplification of the hbb gene, or by direct sequencing of the PCR amplicon generated from the rrs (16S)-rrl (23S) intergenetic spacer. The most prevalent B. burgdorferi genospecies identified were B. afzelii (61.6%), followed by B. garinii (23.4%) and B. burgdorferi sensu stricto (10.6%). B. valaisiana (4.5%) was identified in Norwegian ticks for the first time. Mixed infections were observed in 0.3% of the infected ticks. A higher prevalence of B. burgdorferi s.l. was found in the present study than what has been reported in previous Nordic studies.