Genetic data can help elucidate the dynamics of biological invasions, which are fueled by the constant expansion of international trade. The introduction of European gypsy moth (Lymantria dispar dispar) into North America is a classic example of human-aided invasion that has caused tremendous damage to North American temperate forests. Recently, the even more destructive Asian gypsy moth (mainly L. d. asiatica and L. d. japonica) has been intercepted in North America, mostly transported by cargo ships. To track invasion pathways, we developed a diagnostic panel of 60 DNA loci (55 nuclear and 5 mitochondrial) to characterize worldwide genetic differentiation within L. dispar and its sister species L. umbrosa. Hierarchical analyses supported strong differentiation and recovered five geographic groups that correspond to 1) North America, 2) Europe plus North Africa and Middle East, 3) the Urals, Central Asia, and Russian Siberia, 4) continental East Asia, and 5) the Japanese islands. Interestingly, L. umbrosa was grouped with L. d. japonica, and the introduced North American population exhibits remarkable distinctiveness from contemporary European counterparts. Each geographic group, with the exception of North America, shows additional lower-level structures when analyzed individually, which provided the basis for inference of the origin of invasive specimens. Two assignment approaches consistently identified a coastal area of continental East Asia as the major source for Asian invasion during 2014–2015, with Japan being another source. By analyzing simulation and laboratory crosses, we further provided evidence for the occurrence of natural Asian-North American hybrids in the Pacific Northwest, raising concerns for introgression of Asian alleles that may accelerate range expansion of gypsy moth in North America. Our study demonstrates how genetic data contribute to bio-surveillance of invasive species with results that can inform regulatory management and reduce the frequency of trade-associated invasions.