Objective  To explore the operative strategy of resection of gliomas in cerebral midline area and conduct the molecular biological analysis. Methods  Data of 28 patients with glioma in cerebral midline area who underwent resection under microscope with the assistance of neuroendoscopy if necessary were analyzed. Tumor samples were stored after operation IDH1/2 mutation and MGMT promoter methylation were detected by PCR. The survival time of all patients was statistically analyzed by Kaplan-Meier analysis. Results  Histopathological examination after operation revealed that 2 patients with pilocytic glioma (7%), 11 patients with WHO grade Ⅱ glioma (39%), 5 patients with WHO grade Ⅲ glioma (19%), and 10 patients with WHO grade Ⅳ glioma (35%). Kaplan-Meier analysis indicated that the mean overall survival of all patients was 15 months. The mean overall survivals of patients with low grade glioma and high grade glioma were 26 and 9 months, respectively, and the difference was statistically significant (P<0.01). PCR analysis of tumor samples after operation showed that only 3 patients with positive mutation of IDH1/2 (11%) and 4 patients with positive MGMT promoter methylation (14%). Conclusion  Combination of various technologies such as neuroendoscopy and intra-operative navigation is a major method to improve the prognosis of patients with glioma in cerebral midline area.