Adult female mosquitoes within the subgenus Culex (Melanoconion) Theobald (Diptera: Culicidae) are difficult to identify to species using external morphological features. We present two multiplexed polymerase chain reaction assays that quickly and accurately identify specimens from the southeastern United States based on sequence differences in the internal transcribed spacers of the ribosomal DNA gene array. One assay identifies all species that occur only in Florida, whereas the second assay identifies species that may occur in other southeastern states. These assays require small amounts of DNA, such as DNA from two sonicated legs, or an individual specimen. These assays also may be run as multiple singleplex reactions to determine the mosquito species composition of virus-positive mosquito pools. Reaction volumes may be as low as 10 µl, which reduces assay cost.