The small hive beetle, Aethina tumida Murray (Coleoptera: Nitidulidae), is a serious threat to the honey bee industry, which relies on chemicals for the control of major honey bee pests. We developed a glass vial bioassay for resistance monitoring of adult A. tumida populations in honey bee colonies. We also determined concentrations that could be used to discriminate between susceptible and resistant strains. These include the pyrethroids cypermethrin (25.0 µg per vial) and fluvalinate (25.0 µg per vial) and the organophosphates malathion (10.0 µg per vial), chlorpyrifos (2.5 µg per vial), and coumaphos (25.0 µg per vial). Here, we report that resistance to fluvalinate and coumaphos was widespread in A. tumida populations in Florida in 2019. Aethina tumida populations were still susceptible to cypermethrin, malathion, and chlorpyrifos. The levels of resistance differed between pyrethroid and organophosphate insecticides. Over the last 10 yr, A. tumida populations have developed 43.7-fold resistance to coumaphos and 5.4-fold to fluvalinate. The levels of insecticide resistance were not similar within insecticides in the same class, which suggest that this type of resistance is manageable. Our results demonstrate the usefulness of glass vial bioassays to detect resistance in adult A. tumida and provide the foundation for a resistance management strategy. To the best of our knowledge, this is the first report of insecticide resistance in small hive beetle populations and suggests an urgent need for alternative control strategies for these serious pests of honey bee colonies.