Methylation of the promoter region of the O ( 6 ) -methylguanine-DNA methyltransferase (MGMT) gene is known to be predictive of response to temozolomide treatment in patients with glioblastoma. Contrastingly, little is known about variation in the methylation status of the MGMT promoter after treatment or across different regions of the same tumor. About 22 samples from 10 patients who had undergone multiple resections of a glioblastoma were examined with promoter sequencing. Of these, 20 were also analyzed using Methylation Specific PCR (MSP). The methylation status of the MGMT promoter was altered in the specimens obtained pre and post treatment in 2 of 9 samples as assessed by MSP and 7 out of 10 patients as assessed by promoter sequencing. In four patients, the MGMT promoter was unmethylated at primary surgery, but displayed some methylation (32, 44, 12, and 4%) on post-treatment sampling. Alteration in MSP status from unmethylated to methylated was also observed in 2 of these 4 patients. In another patient, methylation increased from 40% on initial sampling to 68% on the second sample. The remaining two patients initially demonstrated some degree of methylation (72% and 12%); subsequent sampling showed no methylation of the MGMT promoter. To ensure variable methylation status was not due to intra-tumoral variability, three to four specimens were sampled from different regions of large glioblastomas (n = 7). Promoter sequencing revealed minimal variation in methylation in all but two sites examined. Immunohistochemistry also demonstrated minimal change in MGMT expression across the tumors. This suggests that variation in MGMT promoter methylation can occur within the same tumor after treatment, necessitating caution in clinical decision-making based on this analysis.