Canine dirofilariasis caused by Dirofilaria immitis is usually diagnosed by specific antigen testing and/or identification of microfilariae. However, D. immitis and at least six other filariae can produce canine microfilaremias with negative heartworm antigen tests. Discriminating these can be of clinical importance. To resolve discordant diagnoses by two diagnostic laboratories in an antigen-negative, microfilaremic dog recently imported into the US from Europe we developed a simple molecular method of identifying different microfilariae, and subsequently validated our method against six different filariae known to infect dogs by amplifying ribosomal DNA spacer sequences by polymerase chain reaction using common and species-specific primers, and sequencing the products to confirm the genotype of the filariae. We identified the filaria in this dog as D. repens. This is the first case of D. repens infection in the United States. Additionally, we examined microfilariae from five additional antigen-negative, microfilaremic dogs and successfully identified the infecting parasite in each case. Our diagnoses differed from the initial morphological diagnosis in three of these cases, demonstrating the inaccuracy of morphological diagnosis. In each case, microfilariae identified morphologically as A. reconditum were identified as D. immitis by molecular methods. Finally, we demonstrated that our PCR method should amplify DNA from at least two additional filariae (Onchocerca and Mansonella), suggesting that this method may be suitable for genotyping all members of the family Onchocercidae.