Abstract Ultra high performance liquid chromatography-electrospray ionization-mass spectrometry (UHPLC-ESI-MS) technique combined with multivariate and univariate data analysis methods was used to analyze lipid metabolomics of mitochondria-damaged Saccharomyces cerevisiae (ResD-4) after heavy ion beam radiation. The composition of lipid metabolites in Control and ResD-4 was detected, and a total of 648 kinds of lipid molecules were identified. The results showed that the orthogonal partial least squares discriminant analysis (OPLS-DA) could accurately distinguish the lipid metabolites of ResD-4 and Control. On the basis of variable importance for the projection (VIP), variation multiple (FC) and difference analysis (p), 12 subfamilies, 54 types of significant difference in lipid molecules were finally screened including DG (16:0/18:1), LPC (18:1), LPE (16:0), LPI (18:1), LPS (18:1), PA (16:0/18:1), PC (31:1), PE (16:0/10:0), PI (15:0/18:1), PS (16:0/16:1), SQDG (37:4), TG (15:0/16:0/18:1), etc. Significant differences in the expression level of lipid molecules showed that the expression levels of DG, TG, SQDG, PA, PC, PI, LPC, LPE and LPI were up-regulated, and PE, PS and LPS were down-regulated. The results showed that S. cerevisiae might respond by adjusting the content of lipid components after mitochondrial damage caused by heavy ion beam radiation. The lipid molecules with significant difference expression level might be potential markers for S. cerevisiae mitochondrial damage induced by heavy ion beam radiation.