Species of the heteropteran subfamily Lygaeinae possess special subcuticular compartments to store cardiac glycosides, plant‐derived defensive compounds, which they release upon predator attack. In adults of the large milkweed bug, Oncopeltus fasciatus, these storage compartments have previously been described as a modified integument, forming a fluid‐filled dorsolateral space. Here we use three‐dimensional imaging of serial histological sections and synchrotron radiation‐based micro‐computed tomography data to reveal the morphology of these storage compartments and the mechanisms used for the release of a cardiac glycoside‐rich fluid upon attack. Our comparative analysis revealed that the morphology and release mechanism vary among the species investigated. By reconstructing these traits on a recent molecular phylogeny of the Lygaeinae, we demonstrate that the adaptations for the storage and release of cardiac glycosides have evolved in a stepwise manner.