Aedes triseriatus is the principal vector of La Crosse virus (LACv), which is the most common cause of pediatric arboviral encephalitis in North America. Here we report a novel species-specific polymerase chain reaction (PCR) assay that differentially identifies Ae. triseriatus and Ae. hendersoni. Because these 2 sibling species differ in their abilities to transmit LACv, accurate identification is critical for surveillance, research, and control programs. This duplex assay can detect the presence of both species in a single PCR reaction and is therefore simpler and faster than previously reported methods.