Lyme borreliosis (LB) is the most frequent human tick-borne disease in Scandinavia. This master thesis is focusing on the Borrelia burgdorferi sensu lato (s.l.) complex, which is the causative agent of LB. Today, the B. burgdorferi s.l. complex consists of 20 different genotypes, in which three (B. afzelii, B. garinii and B. burgdorferi sensu stricto) are commonly known as human pathogens. The ticks may be infected when they feed of an infected animal, and the spirochete may be further transferred when the vector feeds of the next host. During the past decades, the abundance of the main tick vector, Ixodes ricinus in Scandinavia, seems to have increased due to factors as increased roe deer abundance, changes in habitat structure and climatic factors. This master thesis describes the prevalence and genotype composition of B. burgdorferi s.l. in host-seeking I. ricinus ticks at different locations in Scandinavia. The locations in Norway were Hillevågen (n=100), Håøya (n=100), Tromøya (n=100), Brønnøya (n=98) and Spjærøya (n=70). One location was included from Denmark (Tokkekøb Hegn (n=100)), whereas ticks from two nearby locations in Sweden (Verkö (n=90) and Aspö (n=10)) were included. A total of 668 host-seeking nymphs were investigated for infection with B. burgdorferi s.l. by real-time polymerase chain reaction (PCR) amplification of the 16S rRNA gene. Borrelia spp. were genotyped by melting curve analysis after real-time PCR amplification of the hbb gene, as well as direct sequencing of the rrs (16S)-rrlA (23S) intergenic spacer. The second aim of this thesis was to compare tick DNA extraction methods, and study if these DNA extraction methods influence detection of B. burgdorferi s.l. DNA. In August of 2013, host-seeking I. ricinus nymphs were collected from Hummervika in Søgne, and DNA was extracted by four different methods (n=100 in each method); DNeasy Blood and Tissue Kit (Qiagen), phenol-chloroform, the Abbott m2000sp machine and the NukEx Kit (Orion Diagostica). In addition, samples were analyzed for B. burgdorferi s.l. infection as described above.