European gypsy moth populations (Lymantria dispar L.) are well established and a proven destructive force in hardwood trees throughout the United States and Canada. Introduction of the exotic Asian gypsy moth into North America would be even more impactful, as Asian gypsy moth populations have wider host ranges, and are capable of naturally dispersing more rapidly due to female flight ability. To support early detection and exclusion of Asian gypsy moth, the U.S. Department of Agriculture (USDA) uses molecular techniques to screen moths trapped in North America for evidence of common Asian genotype. In order to strengthen U.S. domestic capacity to screen moths quickly and efficiently, we report a real-time PCR assay for this pest. A probe system using TaqMan 5' nuclease chemistry is reported for detection of an allele associated with common Asian gypsy moth genotypes. The targeted allele is located at the nuclear FS1 locus currently used by the USDA in conventional PCR tests to screen for evidence of Asian gypsy moth introductions or introgression. The diagnostic probe is successfully multiplexed with a conserved 18S probe system to detect reaction failure due to poor sample quality or quantity. The specificity, sensitivity, and repeatability of the FS1-18S multiplex real-time PCR assay were tested on laboratory-reared and field-collected moths to demonstrate diagnostic utility. Implications of the new assay as a screening tool for evidence of Asian gypsy moth introgression and introduction are discussed.