Background: Hymenoptera that mass-provision their offspring have evolved elaborate antimicrobial strategies to ward off fungal infestation of the highly nutritive larval food. Females of the Afro-European Philanthus triangulum and the South American Trachypus elongatus (Crabronidae, Philanthinae) embalm their prey, paralyzed bees, with a secretion from a complex postpharyngeal gland (PPG). This coating consists of mainly unsaturated hydrocarbons and reduces water accumulation on the prey's surface, thus rendering it unfavorable for fungal growth. Here we (1) investigated whether a North American Philanthus species also employs prey embalming and (2) assessed the occurrence and morphology of a PPG among females of the subfamily Philanthinae in order to elucidate the evolution of prey embalming as an antimicrobial strategy. Results: We provide clear evidence that females of the North American Philanthus gibbosus possess large PPGs and embalm their prey. The comparative analyses of 26 species from six genera of the Philanthinae, using histological methods and 3D-reconstructions, revealed pronounced differences in gland morphology within the subfamily. A formal statistical analysis based on defined characters of the glands confirmed that while all members of the derived tribe Philanthini have large and complex PPGs, species of the two more basal tribes, Cercerini and Aphilanthopsini, possess simple and comparatively small glands. According to an ancestral state reconstruction, the complex PPG most likely evolved in the last common ancestor of the Philanthini, thus representing an autapomorphy of this tribe. Conclusion: Prey embalming, as described for P. triangulum and T. elongatus, and now also for P. gibbosus, most probably requires a complex PPG. Hence, the morphology and size of the PPG may allow for inferences about the origin and distribution of the prey embalming behavior within the Philanthinae. Based on our results, we suggest that prey embalming has evolved as an antimicrobial strategy in and is restricted to the tribe Philanthini, which seems to face exceptional threats with regard to fungal infestations of their larval provisions.