Dirofilaria immitis is the causative agent of heartworm disease in canines and felines, and pulmonary dirofilariasis in man. It harbors a symbiotic intracellular bacterium from the genus Wolbachia that plays an important role in its biology and contributes to the inflammatory pathology of the heartworm. This endosymbiont is sensitive to the tetracycline family of antibiotics prompting its use in the treatment of filariasis. To track Wolbachia during treatment, primers were designed based on the FtsZ gene from Wolbachia. These primers amplify a single PCR product with the expected size from DNA samples derived from various species of worms that harbor Wolbachia (D. immitis, Brugia malayi and Brugia pahangy). The detection limit of Wolbachia DNA in the assay was 80 pg of D. immitis DNA. Furthermore, the primer set successfully amplified the expected PCR product using blood samples from dogs harboring the heartworm and circulating microfilariae.