Smoking-related diseases remain a significant public health concern, and heated tobacco products (HTPs) have emerged as a potential alternative to cigarettes. While several studies have confirmed that HTP aerosols contain lower levels of harmful and potentially harmful constituents (HPHCs) than cigarette smoke, less is known about constituents that are intrinsically higher in HTP aerosols. This study provides a comprehensive comparative assessment of an HTP aerosol produced with Tobacco Heating System 2.2 (THS) and comparator cigarette (CC) smoke aiming at identifying all unique or increased compounds in THS aerosol by applying a broad set of LC–MS and GC × GC–MS methods. To focus on differences due to heating versus burning tobacco, confounding factors were minimized by using the same tobacco in both test items and not adding flavorants. Of all analytical features, only 3.5%—corresponding to 31 distinctive compounds—were significantly more abundant in THS aerosol than in CC smoke. A notable subset of these compounds was identified as reaction products of glycerol. The only compound unique to THS aerosol was traced back to its presence in a non-tobacco material in the test item and not a direct product of heating tobacco. Our results demonstrate that heating a glycerol-containing tobacco substrate to the temperatures applied in THS does not introduce new compounds in the resulting aerosol compared to CC smoke which are detectable with the method portfolio applied in this study. Overall, this study contributes to a better understanding of the chemical composition of HTP aerosols and their potential impact on human health.