This study describes the use of a newly constructed set of primers that amplifies an 85-base pair (bp) segment of Borrelia burgdorferi chromosomal DNA. This 85-bp product is not produced when other Borrelia species, Leptospira, or other bacteria are subjected to polymerase chain reaction (PCR). We also describe a rapid method of optimizing the amplification of B. burgdorferi DNA from canine ethylenediaminetetraacetic acid-treated blood and urine samples that circumvents some of the problems encountered due to low number of spirochetes in clinical specimens and that removes inhibiting substances, which improves the PCR diagnosis of canine Lyme borreliosis.