Diapause in insects occurs in response to environmental cues, such as changes in photoperiod, and it is a major adaptation by which insects synchronize their activity with biotic resources and environmental constraints. For multivoltine agricultural insect pests, diapause initiation is an important consideration in management decisions, particularly toward the end of the growing season. The grape berry moth, Paralobesia viteana (Clemens), is the main insect pest affecting viticulture, and this insect responds to postsummer solstice photoperiods to initiate diapause. Because the range of grape berry moth extends from southern Canada to the southern United States, different populations are exposed to different photoperiodic regimes. We quantified the diapause response in grape berry moth populations from Arkansas, Michigan, New York, Pennsylvania, Texas, and Virginia, and observed latitudinal variation in diapause initiation. Populations from Michigan, New York, and Pennsylvania responded significantly different than those from Arkansas, Texas, and Virginia. We also observed, as a consequence of our experiments, that the timing of our laboratory studies influenced grape berry moth's response to photoperiod, ceteris paribus. Experiments that were conducted when grape berry moth would be naturally in diapause resulted in a significant higher proportion of diapausing pupae at photoperiods (i.e., > 15 h) that generally do not induce diapause, suggesting that attention should be paid to the timing of behavioral and physiological experiments on insects. This relationship between photoperiod and diapause induction in grape berry moth across geographic regions will provide applicable knowledge to improve pest management decisions.