The potato tuberworm, Phthorimaea operculella Zeller, is one of the most damaging pests of potatoes worldwide. Although tuberworm was recorded in California as early as 1856, it was first reported in damaging numbers in Oregon and Washington in the early 2000s. The objective of this study was to provide baseline information on the population structure of potato tuberworm in the United States. Potato tuberworm adults were collected from potato fields in the major production regions in the United States. Amplified fragment length polymorphisms (AFLP) were used to determine the genetic population structure of potato tuberworm. We used 219 individuals and 335 polymorphic AFLP bands to infer the geographic population structure of potato tuberworm. Posterior probabilities calculated using the model-based clustering method implemented in STRUCTURE, and Fst values calculated using AFLP-SURV confirmed the existence of two distinct populations in the United States (one mostly associated to the western United States and the other one mostly associated to the eastern United States). This study provides baseline data for the molecular characterization of potato tuberworm populations, which will aid in tracking the origin of future invasions within the United States.