Abstract Objective The limitations of histopathology in the diagnosis of glioblastoma were analyzed by comparing histopathological and molecular pathological results in patients with glioblastoma.Methods The pathological data of 91 patients with glioblastoma who underwent surgical treatment in the Neurosurgery Department of The Affiliated Hospital of Xuzhou Medical University from January 2020 to May 2022 were retrospectively analyzed. With molecular pathology as the gold standard, patients whose histopathology was inconsistent with molecular pathology in the diagnosis of glioblastoma were classified as group A (30 cases)，the patients who were consistent were classified as group B (61 cases), and the limitations of histopathology in the diagnosis of glioblastoma were analyzed by comparing the specific differences between the two groups.Results The consistency rate between histopathology and molecular pathology in the diagnosis of glioblastoma was only 67.03%, and the consistency rate between IDH-1 mutation, MGMT promoter methylation and ATRX mutation and molecular pathology were 84.62%, 59.34% and 83.51%, respectively.The histopathological accuracy of IDH-1 was significantly different between the two groups (66.66% vs 93.44%, P < 0.05), and the expression index of Ki-67 was also significantly different between the two groups (P < 0.001).The histopathology of the two groups showed no significant difference in the accuracy of MGMT methylation and ATRX mutation.Conclusion Histopathological detection accuracy of MGMT methylation in patients with glioblastoma was low.Histopathological errors occurred in the diagnosis of glioblastoma, mainly in idH-1.For glioma patients with low KI-67 index but rapid clinical progression, molecular pathological detection should also be performed.