The larvae of the diamondback moth, Plutella xylostella, cause major economic losses to cruciferous crops, including cabbage, which is an important vegetable crop in China. In this study, we used the mitochondrial COI gene and 11 ISSR markers to characterize the genetic structure and seasonal migration routes of 23 P. xylostella populations in China. Both the mitochondrial and nuclear markers revealed high haplotype diversity and gene flow among the populations, although some degree of genetic isolation was evident between the populations of Hainan Island and other sampling sites. The dominant haplotypes, LX1 and LX2, differed significantly from all other haplotypes both in terms of the number of individuals with those haplotypes and their distributions. Haplotypes that were shared among populations revealed that P. xylostella migrates from the lower reaches of the Yangtze River to northern China and then to northeastern China. Our results also revealed another potential migration route for P. xylostella, i.e., from southwestern China to both northwestern and southern China.