Acarapis woodi Rennie (Acari: Tarsonemidae) is an endoparasitic mite, which can affect the respiratory system in the honey bee Apis mellifera L. (Hymenoptera: Apidae) causing mortality. This mite was first recorded in Brazil in the 1970s, but its current presence is unclear. Therefore, we evaluated the presence of A. woodi in the Africanized honey bee A. mellifera in Brazil, to update the occurrence data of this parasite, 47 yr after its detection. We examined a total of 153 honey bee colonies, from 15 different states using dissection and molecular techniques. This is the first study of the detection of A. woodi in Brazil using molecular techniques. The results were negative for both methods employed, and we can conclude that A. woodi is not present in Africanized honey bee colonies in Brazil.