Trichoplusia ni is a subtropical moth that migrates annually from southern California to southern British Columbia, Canada where it invades vegetable greenhouses and field crops. The heated greenhouse environment has altered the natural extinction–recolonization dynamics of T. ni populations, and allows year-round persistence in some locations. In addition, the extensive use of the biopesticide, Bacillus thuringiensis subspecies kurstaki (Bt) in some greenhouses has selected for resistance. Here we investigated the genetic structure of T. ni populations in British Columbia greenhouses and in field populations in California and British Columbia using amplified fragment length polymorphisms (AFLP) as related to patterns of Bt resistance. The majority of British Columbia field populations were similar to the California field populations, the potential source of migrants. However populations in two geographic areas with high concentrations of greenhouses showed local genetic differentiation. Some of these populations experienced severe bottlenecks over-winter and following Bt sprays. Greenhouse populations showed a pattern of isolation by distance and a strong positive relationship between genetic differentiation and levels of Bt resistance. These patterns indicate that greenhouses that sometimes support year-round populations of T. ni and the ensuing strong bottlenecking effects following winter cleanups and Bt application cause genetic differentiation of T. ni populations. Long distance migrants to field populations contribute to genetic homogeneity of these.