AIM: To evaluate the overlapping and distinguishing cytologic features of primary central nervous system lymphoma (PCNSL), diffuse large B-cell lymphoma, and glioblastoma (GM) in frozen sections and squash smear slides. MATERIALS AND METHODS: Intraoperative frozen sections and squash smear slides from PCNSL (N = 63) and GM (N = 122) patients diagnosed from 2005 to 2015 were retrieved from pathology records. Overlapping and distinguishing histologic features were examined and statistically analyzed. RESULTS: Necrosis and moderate nuclear size variation were common features of PCNSL and GM. PCNSL characteristically showed apoptosis, lack of a fibrillary background, monotonous nuclei, scant cytoplasm, lack of microvascular proliferation, and presence of perivascular cuffing. Multivariate analysis revealed that presence of apoptosis was the most powerful predictive parameter for the diagnosis of PCNSL. CONCLUSION: The presence of apoptosis was effective for the intraoperative diagnosis of PCNSL compared to GM..