Varroa destructor Anderson & Trueman (Parasitiformes: Varroidae) is an ectoparasitic pest of the western honey bee (Apis mellifera L.; Hymenoptera: Apidae) colonies. The ability to study all life stages of the mite in a laboratory setting requires one to rear the mite in vitro. This is a crucial step for the advancement in research studies, and the development of management protocols for Varroa. Current practices require that Varroa be collected from field colonies for use in lab-based studies. Traditional collection techniques for obtaining mites from adult bees include using carbon dioxide or a method in which a combination of powdered sugar and shaking dislodges the mites from a group of adult bees (i.e., a “sugar shake”). Herein, we compared 2 mite collection techniques and measured mortality of the mites after collection using the Varroa maintenance system, a tool for maintaining in vitro populations of Varroa on their host. Our results indicate that mites collected using the sugar shake method lived significantly longer (> 6 d, with 20% mortality at 6 d) than did those collected using carbon dioxide (3.9 d, with 66% mortality at 6 d). Carbon dioxide exposure was detrimental to the recovery of Varroa. These data provide critical information on how to collect Varroa properly for use in in vitro survival studies.