Background The purpose of this study was to evaluate the ability of intraoperative ultrasound (IOUS) to differentiate the gross pathological features of brain mass lesions in comparison with preoperative imaging and confirmable histopathological results. Results A total of 365 patients were operated on for brain mass lesions removal from May 2017 to May 2022 under the guidance of intraoperative ultrasound with transducers 2.5–8 megahertz (MHZ). Ultrasound gross pathological findings were compared to the preoperative imaging and the confirmable histopathological results. Intraoperative ultrasound defined either internal or external gross pathological features of all brain mass lesions. The IOUS showed spontaneous enhancements of the brain abscess walls, which were equivalent to contrasted CT and MRI. Significantly large diameters were noted in the IOUS measurement of abscesses in comparison with CT and MRI (P = 0.001). The walls of the brain abscesses were significantly well defined in IOUS imaging in comparison with CT (P = 0.001) and equivalent to MRI. IOUS showed equivalent significance to CT and MRI in characterizing intra-parenchymal hematomas. Significantly large diameters were noted in the IOUS measurement of hematomas in comparison with CT and MRI (P = 0.001). IOUS showed significant definition of brain tumors in comparison with CT and MRI regarding tumor edge definition, tumor contours, necrosis, and cystic components (cystic definition, cystic multiplicity, cystic trabeculations, and cystic wall thickness) (P = 0.001). IOUS was equivalent to CT and MRI regarding intratumoral hematomas and brain edema. IOUS was equivalent to CT regarding calcification detection. The significant criteria for high-grade brain tumors versus low grade by IOUS were: P = 0.001 (necrosis, brain edema, rare calcifications, presence of cystic components, thick cystic walls, large diameter, hypo-echogenicity, and heterogeneity); P = 0.002 (cystic trabeculations); P = 0.005 (multiple cysts); and P = 0.03 (irregular contour). IOUS can characterize brain tumors and suspect specific and significant criteria for many types with great overlap. Conclusions Intraoperative ultrasound has the ability to differentiate the gross pathological features of brain mass lesions in comparison with preoperative imaging and confirmable histopathological results.