Technical Abstract: The tracheal mite, Acarapis woodi, has become a worldwide pest of honey bees. Identification and description of A. woodi is hampered by the minute size of these mites, their cryptic lifestyle, and a general lack of strong morphological differences with relatives. Here we describe a diagnostic genetic test for these mites, based on the mitochondrial cytochrome oxidase I gene. The test is highly specific and sensitive, on par with expert dissections of honey bee trachea. We further describe a nested-PCR approach that is capable of detecting even single mites. This diagnostic test can complement and expand upon existing methods and offers a new opportunity to explore taxonomic and population-genetic questions in tracheal mites.