Abstract: Sugar esters in tobacco play an important role as precursors generating tobacco aroma species, and their presence significantly influence the quality of tobacco leaves. Typically present in trace amounts, sugar esters in tobacco exhibit a diverse range of isomers, making their separation and analysis a significant challenge. To address this challenge, mass spectrometry has been extensively applied for the identification and differentiation of various of sugar ester compounds in tobacco. However, there is no study regarding the separation and analysis of sugar esters specifically within Burley tobacco leaves. In this research, Burley tobacco leaf extracts were obtained using the solvent extraction technique, and underwent separation using silica gel column chromatography. Ultimately, the fractions comprising glucose esters and various sucrose esters were collected, respectively. The structural identification of these acquired sugar esters was achieved by liquid chromatography-tandem mass spectrometry (LC-MSⁿ). According to the MS² fragment ions of sucrose esters, the mass difference of 162.05 and 204.06 was mainly observed, implying an unbound fructose ring and a fructose ring acetylated in the sucrose ester, respectively. The composition information of fatty acids substituted to the glucose ring were further revealed based on the MS³ or MS² fragment ions from sucrose esters or glucose esters. By combining the relative molecular weight, molecular formula, and the above mentioned fragment information, four types of sucrose esters from SE-I to SE-IV (referred to as compound 1-20) and two types of glucose esters GE-I to GE-II (referred to as compound 21-31) were finally identified. Among them, four sucrose esters of distinct molecular weights were newly discovered, and the structures of eight glucose esters were reported for the first time in cultivated tobacco. The sugar esters were analyzed by high-resolution mass spectrometry, which could effectively distinguish the number of fatty acids substituted on glucose and fructose, thus facilitating the differentiation of sugar ester types. Nevertheless, the identification of sugar esters in this study was limited to determine the characteristic skeleton and fatty acid composition. Detailed information on specific fatty acid types and substitution sites require further investigation using NMR technique. In conclusion, these results contribute to the current knowledge of sugar esters in Burley tobacco by expanding the range of identified types and providing high-resolution mass spectrometry data for various types of sugar esters for the first time. These findings hold significant reference value for the analysis, identification, and metabolism of different sucrose and glucose esters in tobacco.