O6-methylguanine-DNA methyltransferase (MGMT) is a repair enzyme that removes promutagenic O6-methylguanine adducts in DNA, to protect cells from acquisition of G:C→ A:T mutations. MGMT promoter methylation and polymorphisms may affect MGMT expression and activity. In the present study, we assessed MGMT promoter methylation and polymorphisms (Leu84Phe, Ile143Val, c.–56C>T) in 371 glioblastomas diagnosed at the population level. MGMT methylation was observed in 165 (44%) glioblastomas, with a higher frequency in females than males (53 vs. 39%; p = 0.0106) and in secondary than primary glioblastomas (73 vs. 43%; p = 0.0074). The frequency of TP53 G:C→A:T mutations in glioblastomas with MGMT methylation was 25%, which was significantly higher than that in glioblastomas with MGMT methylation (16%; Fisher exact test; p = 0.0385). MGMT 143 Val allele in glioblastomas was significantly less frequent than in a healthy European Caucasian population, and was associated with longer survival than those with the MGMT 143 Ile allele (hazard ratio 0.70; 95% CI 0.48–1.01). These results suggest that MGMT methylation may be associated with susceptibility to acquire TP53 G:C→A:T mutations, and that MGMT polymorphisms may affect the risk and prognosis of glioblastomas.