To confirm the substances that make adlay seeds smelly, the volatiles of adlay seeds (Coix lachryma-jobivar. ma-yuen Stapf) of three off-flavor levels were studied. For the analysis, the headspace-solid phase micro-extraction (HS-SPME) method was used in combination with gas chromatography-mass spectrometry (GC-MS) and GC–olfactometry (GC-O), where the human nose is used as a sensitive and specific detector for off-flavor compounds. More than 79 of volatile compounds which belonged to distinct chemical families were analysed. A total of 47, 43 and 51 volatile compounds were identified in fresh, faintly smelly and strongly smelly adlay seeds with total relative contents of 96.14%, 98.68% and 98.00%, respectively. Meanwhile, 12 kinds of off-flavor active compounds were detected from the strong smelly adlay seed volatiles by GC-O and odor activity value method (OAV), including hexanal, heptanal, 2-heptenal, n-octanal, (E)-2-octenal, 1-nonanal, (E)-2-nonenal, decanal, 2-decenal, hexanoic acid, 2-nonanone and 6-undecanone.