The purpose of this study was to determine the prevalence of Lyme Disease (LD) bacteria in adult Ixodes pacificus ticks collected from the mountains of San Bernardino County in Southern California. Seven hundred fifty four I. pacificus adults were collected from the Pacific Crest Trail and adjacent areas. The Polymerase Chain Reaction (PCR) was used to screen ticks for Borrelia burgdorferi infection by targeting two different DNA loci. Oligonucleotide primers targeting both the ospA and fla genes were used in the assay. Ticks were processed in pools of three, and genomic DNA from the ticks was extracted with a commercial mini-kit utilizing silica matrix spin-columns. All ticks tested negative for B. burgdorferi infection regardless of primer pair used. In addition, ticks were negative following examination by dark-field microscopy. This study confirms previous reports that the prevalence of LD in Southern California is quite low.