Eusocial insects exhibit reproductive division of labour, in which one or a few queens perform almost all of the reproduction, while the workers are largely sterile and assist in rearing their siblings. Consequently, many of the colony’s tasks (e.g. nest construction and brood rearing) should be modulated depending on whether the queen is fertile. Here, we tested whether queens’ cuticular lipids could provide reliable signals of fertility in the honeybee Apis mellifera, as they do in other social Hymenoptera. Specifically, we tested whether cuticular lipids differ between virgin queens of different ages, and between queens exposed to different artificial insemination treatments being semen (sperm and seminal fluid), seminal fluid only or saline control. Using gas chromatography–mass spectrometry, we found 27 lipids: 21 different hydrocarbons, namely alkanes, alkenes and dienes, as well as six wax esters. The lipid profile changed dramatically in the first 10 days after eclosion, and there were differences in lipid composition between virgin and artificially inseminated queens. Insemination with semen, seminal fluid or saline did not result in distinct chemical profiles. Our findings indicate that the physical stimulus of insemination was responsible for the observed changes in the cuticular profile in honeybee queens. Our results demonstrate that cuticular lipid profiles encode information on queen age, fertility and mating status, which could in principle be utilised by workers and rival queens.