Temozolomide (TMZ) is considered a first line chemotherapy drug for glioblastoma (GBM). Unfortunately, the GBM without methylation of O6-methylguanine-DNA methyltransferase (MGMT), accounting for about 70% of all GBM, shows an inherent resistance to TMZ treatment. Aberrant accumulation of neutral lipids, primarily triglycerides (TGs) and cholesteryl esters (CEs), in lipid droplets (LDs) has been recognized as metabolic vulnerability for GBM therapy. However, it is not known whether MGMT methylation affects lipid accumulation in GBM. Herein, we employed label-free Raman spectromicroscopy, which integrated stimulated Raman scattering (SRS) microscopy and confocal Raman spectroscopy, to quantitatively analyze both the amount and composition of intracellular LDs in intact GBM tissues obtained from patients who had undergone resection surgery. Our results showed significant reductions in both the LD amount and the CE percentage in MGMT unmethylated GBMs (MGMT methylation < 15%) compared to MGMT methylated ones (MGMT methylation ≥ 15%). Due to a big variation of lipid accumulation in the MGMT methylated GBMs, these patients were further divided into hypermethylated group (MGMT methylation ≥ 50%) and intermediate-methylated group (MGMT methylation 15∼50%), according to the significantly different median survival rates of these two groups. Remarkable differences in LD amount, CE percentage, and also lipid saturation degree were found between the hypermethylated group and the other two groups, but not between the unmethylated and intermediate-methylated groups. To elucidate the possible underlying mechanism, we analyzed the differential expression of lipid metabolism-related genes in GBM with different levels of MGMT methylation using The Cancer Genome Atlas Program (TCGA) dataset. It was shown that the genes related to lipid oxidation and lipid efflux were upregulated, and the genes related to lipid synthesis were downregulated in unmethylated group. These findings unravel the relationship between MGMT methylation and lipid accumulation in GBM, which may offer new opportunities for the diagnosis and treatment of TMZ-resistant GBM.