The occurrence of resistance in Varroa mite populations is a serious threat to the beekeeping industry and to crops that rely on the honey bee for pollination. Integrated pest management strategies for control of this pest include the judicious use of insecticides. To monitor field populations of Varroa mite for insecticide resistance, a glass vial bioassay procedure was developed to use in the development of a resistance management strategy. Diagnostic concentrations needed to separate susceptible genotypes from resistant individuals were determined for Cypermethrin (0.1 µg per vial), fluvalinate (5.0 µg per vial), malathion (0.01 µg per vial), coumaphos (10.0 µg per vial), diazinon (5.0 µg per vial), methomyl (0.5 µg per vial), propoxur (0.1 µg per vial), and endosulfan (2.5 µg per vial). Resistance to organophosphorus insecticides (malathion, coumaphos) and pyrethroids (cypermetrhrin, fluvalinate) was widespread in both La Media Ranch, TX, and Wewahitchka, FL, from 2007 to 2009. There was no resistance to endosulfan, diazinon, methomyl, and propoxur in field populations of Varroa mite in the two locations where resistance was monitored. The seasonal patterns of resistance in Wewahitchka were different from those of La Media Ranch. In the former location, the frequency of resistance to all insecticides tested decreased significantly from 2007 to 2009, whereas it increased in the latter location. Resistance levels were unstable, suggesting that resistance could be successfully managed. The results validate use of the glass vial bioassay to monitor for resistance in Varroa mite and provide the basis for the development of a resistance management strategy designed to extend the efficacy of all classes of insecticides used for control of Varroa mite.