Tissues from Dutch family dogs symptomatic for borreliosis according to established criteria and from infected but asymptomatic dogs were tested forBorrelia burgdorferisensu lato DNA using a polymerase chain reaction. Subsequently,B. burgdorferisensu stricto,B. garinii, B.afzelii, andB.valaisianawere identified by hybridization. Symptomatic dogs showed a higher prevalence ofBorreliain liver samples (9 of 15) than asymptomatic dogs (9 of 43) p= 0.0049. Overall,B. garinii was the most prevalent species and occurred together with up to three other species in one liver sample.B. burgdorferisensu stricto however, was predominantly detected in samples of synovial membranes, skin, cerebrospinal fluid, bladder, heart, and bone marrow. Nine out of 10 symptomatic dogs with a very high antibody titre were positive for Borrelia DNA by PCR in one or more of these tissues. We conclude that dissemination in naturally infected European dogs occurres and that the two most prevalent species,B. burgdorferisensu stricto andB.garinii, differ in their tropism.