Abstract: In the present study, the effects of various saccharification methods on volatile substances in unaged whiskies were investigated. The whisky was prepared using four separate hydrolysis and fermentation methods: double-mash leaching saccharification method (SJ), double mash method with a single boil for saccharification (SY), double mash second boil-out saccharification method (SE), and individual saccharification of raw materials (DD), and two simultaneous saccharification and fermentation processes: simultaneous saccharification and fermentation of clinker (TB) and simultaneous saccharification and fermentation of raw materials (SL). Volatile substances in the unaged whisky were analyzed using electronic nose and headspace solid-phase microextraction (HS-SPME) coupled with comprehensive two-dimensional (2D) gas chromatography quadrupole time-of-flight mass spectrometry. The samples obtained using the separate clinker hydrolysis and fermentation method (TB-J) had significant odor differences from those obtained using the other methods. Furthermore, The contents of sulfur compounds, nitrogen oxides, alcohols, ethers, aldehydes, ketones, and alkanes in TB-J were significantly (P<0.05) lower than those in other methods. The total number of volatile substances detected in the samples from the two simultaneous saccharification and fermentation methods (TB-J (109) and SL-J (114)) was higher than that in the samples from the four separate hydrolysis and fermentation methods (SJ-J (82), SY-J (90), SE-J (92), and DD-J (94)). Three key volatile substances (1-octen-3-ol, linalool, and ethyl isovalerate) were screened between the samples from the simultaneous saccharification and fermentation methods and from the separate hydrolysis and fermentation methods, and a total of 13 differential volatile substances with important effects on flavor were screened. Linalool, trans-nerolidol, hexanoic acid, and 2-phenylethyl ester contents were lower in samples subjected to the simultaneous saccharification and fermentation methods. The contents of the remaining 13 substances (ethyl isovalerate, 2-undecanol, nonanal, 1-octanol, dodecanoic acid, ethyl ester, trans-2-nonenal, 1-nonanol, geranylacetone, 1-decanol, 1-octen-3-ol, hexanal, benzeneacetaldehyde, benzenepropanoic acid, and ethyl ester) were higher in the samples subjected to the simultaneous saccharification and fermentation methods. The findings of this study provide technical support and an informative reference for the use of simultaneous saccharification and fermentation methods for whisky production.