Hymenopteran queens are collectively highly fecund, often long-lived individuals that undergo dramatic physiological changes after they mate and establish a nest. However, the degree to which these changes are conserved among species with different life histories is not well-defined. We conducted a comparative proteomic study investigating differences between reproductive stages (virgins, mated, and established queens) of Apis mellifera, Bombus impatiens, B. terrestris, and Lasius niger. We identified conserved upregulation of proteins involved in anatomical and system development as queens transition to establishing a nest in all species except B. terrestris. We also identified conserved patterns of vitellogenin, vitellogenin receptor, and immune responsive protein (IRP)30, all of which are proteins typically associated with oviposition. However, expression patterns of other immune proteins, heat-shock proteins (HSPs), detoxification enzymes, and antioxidant enzymes were more dissimilar, with some species exhibiting similar trends and coregulation through reproductive stages, while others exhibited variable or opposite patterns. These conserved and unique profiles likely in part reflect similarities and differences in selective pressure on reproductive stages of each species and may indicate differing abilities to respond to emergent pathogens or environmental change.