The two most common causes of canine filariasis are Dirofilaria immitis and D repens, both transmitted by mosquitoes. D immitis is associated with heartworm disease; D repens is localized to subcutaneous tissues and is associated with nodular disease. Both are considered zoonotic diseases, and treatment regimens differ. Differentiation between these two using morphological characteristics and existing ELISA assays can be difficult, especially where both parasites are found. A high-resolution melting (HRM) analysis coupled to real-time PCR was developed. This can be used to target sequence polymorphisms of the same gene in different species without performing DNA sequencing or using species-specific probes. The test was found to reliably discriminate between the two species in single- and mixed-blood samples.