Royal jelly (RJ) is a popular nutraceutical with myriad biological functions. The lipid composition of RJ is unlike most plant and animal materials, giving RJ unique health benefits. In this study, we comprehensively profiled the lipidomes of RJ, via ultra-high pressure liquid chromatography/ion mobility-quadrupole-time-of-flight-mass spectrometry (UHPLC-IM-Q-TOF-MS) combined with gas chromatography-mass spectrometry (GC-MS), and compared the lipid profiles of RJs from different botanical origins. The most abundant macromolecular lipids were phospholipids (4.34–6.62 mg/100 g). Trans-10-hydroxy-2-decenoic acid (10-HAD), which was the main monounsaturated fatty acids (MUFAs), accounted for more than 70% of total fatty acids in RJ, while polyunsaturated fatty acids (PUFAs) content ranged from 96.38 to 125.47 mg/100g. Among the six sterols quantified, 24-methylene cholesterol (24MET) was the most abundant, constituting 29.3%–31.4% of total sterols. Principal component analysis revealed that botanical origin had a pronounced influence on the composition of lipids, especially on phospholipids and low abundance MUFAs. This research will aid in uncovering low abundance lipids in RJ that are beneficial to comprehensively understand the lipid composition of RJ, further evaluate and control RJ quality based on lipid composition.