Wolbachia Hertig and Wolbach (Rickettsiales: Anaplasmataceae) are non-culturable, bacterial endosymbionts that have been found in a broad range of arthropods and other invertebrate species. They have been implicated in human and veterinary pathologies, and may play a major role in embryonic development and evolution of host species. Given the apparent ubiquity of Wolbachia in certain animal taxa suggested by previous studies, there are still many unanswered questions about its biology. Like other obligate intracellular bacteria, they are difficult to cultivate outside of their host and often are analyzed using molecular methods. Polymerase chain reaction (PCR) assays have been developed previously for Wolbachia detection within host species, and several genes have been explored for strain typing and phylogenetic reconstruction. However, given the expansive host range and biological complexity of symbiotic relationships between Wolbachia and its host species, new methods could help accelerate the pace of Wolbachia research. As part of an overarching goal to study the distribution of Wolbachia in local mosquitoes and in the heartworm, Dirofilaria immitis (Leidy) (Rhabditida: Onchocercidae), we aimed to develop cost-effective methods that can be used in strain identification and analysis. We developed a novel PCR assay targeting the gyrA gene of Wolbachia and explored various methods of sample preparation. Presumptive Wolbachia were detected in mosquito specimens from several genera, as well as from D. immitis samples obtained from canine necropsy. DNA sequence analysis of the PCR products confirmed the identity of Wolbachia and revealed variability within some regions of the gyrA gene that correspond to host species. Consequently, this gene could be useful for future phylogenetic and population studies.