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The gypsy moth, Lymantria dispar, is among the most destructive quarantine pests of forests. Here, we reconstructed the genetic structure and determined the population differentiation of gypsy moths across its distribution range at different times. This information could be used to both improve the prevention and detection of gypsy moths in the field. Using 31 newly designed species-specific primers targeting fragments of 216–1102 bp, we identified 103 full-length cytochrome oxidase subunit I (COI) gene sequences from eight fresh samples and 95 L. dispar specimens collected between 1955 and 1996, mainly in China. Combining 103 full-length COI gene sequences with 146 COI gene sequences from Genbank or DNA barcode libraries, we analyzed the genetic differentiation, gene flow and haplotypes between gypsy moth populations in order to reflect the genetic structure and population dynamics of gypsy moths. We discovered 25 previously unknown haplotypes from old gypsy moth specimens. We found that the genetic diversity among gypsy moth populations (collected in the same region at different time points) was relatively high. Furthermore, the genetic structure of Chinese geographical populations (Heilongjiang, Liaoning, Beijing) in different years was distinct. Our results suggested that some gypsy moths in China showed the genetic affinity with European gypsy moths (a sub-species of gypsy moths found mainly in Europe). [ABSTRACT FROM AUTHOR]