Background. The treatment for patients with high-grade gliomas includes radiation therapy and temozolomide. However, some patients do not respond to temozolomide because they have a methylation reversal mechanism through the enzyme O6-methylguanine-DNA-methyltransferase. This biomarker has been used as a prognostic factor in patients receiving treatment with temozolomide. However, not all patients respond in the same way, which suggests the existence of other genes involved in resistance to temozolomide. Materials and Methods. A group of 31 patients with high-grade gliomas was recruited and were clinically, image pattern, and pathologically characterized. The sequencing of 324 genes related to different types of cancer was performed to detect mutations. Subsequently, a statistical analysis was conducted to determine the mutated genes that were most related to resistance to treatment. Results. The genes related to the second relapse of patients with high-grade glioma after the use of temozolomide according to Stupp protocol and metronomic dose were PIK3C2B, KIT, ERBB3, and MLH1. Conclusions. Considering the results obtained, we suggest that the mutations in the four genes and the methylation of the gene promoter that codes for MGMT protein could be related to the clinical evolution of patients with high-grade gliomas and its response to treatment with temozolomide.