Glioma is known with its strong invasion. As there is a limitation in preoperative inspection, the only procedure for improving postoperative result is to examine, during the operation, if there is no residual tumor, and if any, to remove as completely as possible. For this purpose, a quick and compact observation tool with a high contrast and spatial resolution is required. Acoustic micro-imaging for biological tissues can be performed quickly without staining process. This study is aimed for establishing in situ detection for brain tumor during surgery. Under general anesthesia, rats were received with an intracerebral injection of C6 glioma cells. The brain was taken out and its coronal section was exposed 14 days later. The cross section was placed on a polypropylene substrate, and was observed by transmitting an ultrasound from the other side of the substrate. The ultrasound, of which central frequency was ca. 80 MHz, was focused on the interface between the substrate and tissue. The reflection, received by the identical transducer, was interpreted into the local characteristic acoustic impedance (CAI) of the tissue. By scanning the transducer, a cross sectional CAI profile was obtained. The morphological inspection of the CAI profile allowed us to identify the tumor. The average value of CAI in tumor region did not significantly differ from that in the normal region. However, the tumor region showed considerable heterogeneity in the CAI pattern. Standard deviation of CAI in a small probing area was defined as the local heterogeneity. The heterogeneity profile, which was obtained by scanning the probing area, successfully highlighted the tumor region.