The objective of this study was to manipulate ventilation rate to characterize interactions between stocks of honey bees (Apis mellifera L.) and ventilation setting on varroa mite (Varroa destructor Anderson and Trueman) mortality in honey bee colonies kept indoors over winter. The first experiment used colonies established from stock selected locally for wintering performance under exposure to varroa (n=6) and unselected bees (n=6) to assess mite and bee mortality and levels of carbon dioxide (CO₂) and oxygen (O₂) in the bee cluster when kept under a simulated winter condition at 5°C. The second experiment, used colonies from selected bees (n=10) and unselected bees (n=12) that were exposed to either standard ventilation (14.4 liter/min per hive) or restricted ventilation (0.24 liter/min per hive, in a Plexiglas ventilation chamber) during a 16-d treatment period to assess the influence of restricted air flow on winter mortality rates of varroa mites and honey bees. Experiment 2 was repeated in early, mid-, and late winter. The first experiment showed that under unrestricted ventilation with CO₂ concentrations averaging <2% there was no correlation between CO₂ and varroa mite mortality when colonies were placed under low temperature. CO₂ was negatively correlated with O₂ in the bee cluster in both experiments. When ventilation was restricted, mean CO₂ level (3.82±0.31%, range 0.43–8.44%) increased by 200% relative to standard ventilation (1.29±0.31%; range 0.09–5.26%) within the 16-d treatment period. The overall mite mortality rates and the reduction in mean abundance of varroa mite over time was greater under restricted ventilation (37±4.2%) than under standard ventilation (23±4.2%) but not affected by stock of bees during the treatment period. Selected bees showed overall greater mite mortality relative to unselected bees in both experiments. Restricting ventilation increased mite mortality, but did not affect worker bee mortality relative to that for colonies under standard ventilation. Restricted ventilation did not affect the overall level of Nosema compared with the control. However, there was an interaction between stock, season, and time of the trial. Unselected stock showed an increase in Nosema over time in the late winter trial that did not occur in the selected stock. In conclusion, these findings suggested that restricted ventilation has potential to suppress varroa mite in overwintering honey bee colonies via a low-cost and environmentally friendly measure.