AbstractVarroa destructor is considered a major reason for high loss rate of Western honey bee (Apis mellifera) colonies. To prevent colony losses caused by V. destructor it is necessary to actively manage the mite population. Beekeepers, particularly commercial beekeepers, have few alternative treatments other than synthetic acaricides to control the parasite, resulting in intensive treatment regimens that led to the evolution of resistance in mite populations.To investigate the mechanism of the resistance to amitraz detected in V. destructor mites from French and U.S. apiaries, we identified and characterized octopamine and tyramine receptors (the known targets of amitraz) in this species. The comparison of sequences obtained from mites collected from different apiaries with different treatment regimens, showed that the amino acid substitutions N87S or Y215H in the OctβR were associated with treatment failures reported in French or U.S. apiaries, respectively. Based on our findings, we have developed and tested two high throughput diagnostic assays based on TaqMan® able to accurately detect mites carrying the mutations in this receptor. This valuable information may be of help for beekeepers when selecting the most suitable acaricide to manage V. destructor.