Abstract: The investigation focused on the healthy callus of Vaccinium dunalianum, and the methanol content was analyzed using LC−MS technology. The results showed that a total of 281 compounds divided into 14 categories were detected in the positive ion mode, which mainly including 58 terpenoids, 41 alkaloids, 28 organic acids, 24 heterocyclic compounds, 22 glycosides and 13 alcohols, with a relative content of 16.866%, 13.711%, 6.108%, 10.138%, 12.488% and 17.513%, respectively. A total of 71 compounds divided into 13 categories were detected in the negative ion mode, among which 14 alkaloids, 9 organic acids, 9 glycosides, 7 terpenoids and 7 esters were the main components with a relative content of 5.839%, 72.653%, 3.423%, 2.062% and 2.735%, respectively. Especially, the relative content of pyruvic acid was the highest at 47.474%. The results will provide a basis for the further regulation of the major chemical constituents in the callus of V. dunalianum.