ABSTRACTMembers of the genusBorreliaare among the most common infectious agents causing tick-borne disease in humans worldwide. Here, we developed a Light Upon eXtension (LUX) real-time PCR assay that can detect and quantifyBorreliaspecies in ticks that have fed on humans, and we applied the assay to 399 such ticks.BorreliaPCR-positive ticks were identified to species level by sequencing the products of conventional PCR performed usingBorreliagroup-specific primers. There was a 19% prevalence ofBorreliaspp. in the detached ticks, and the number of spirochetes perBorreliaPCR-positive tick ranged from 2.0 × 102to 4.9 × 105, with a median of 7.8 × 103spirochetes. Adult ticks had a significantly larger number of spirochetes, with a median of 8.4 × 104compared to the median of nymphs of 4.4 × 104. Adult ticks also exhibited a higher prevalence ofBorrelia(33%) than nymphs (14%). Among the identified species,Borrelia afzeliiwas found to predominate (61%) and was followed byB.garinii(23%),B.valaisiana(13%),B.burgdorferisensu stricto (1%),B.lusitaniae(1%), andB.miyamotoi-like (1%). Also, 3% of the ticks were coinfected with multiple strains ofB. afzelii. Notably, this is the first report ofB. lusitaniaebeing detected in ticks in Sweden. Our LUX real-time PCR assay proved to be more sensitive than a corresponding TaqMan assay. In conclusion, the novel LUX real-time PCR method is a rapid and sensitive tool for detection and quantification ofBorreliaspp. in ticks.