Abstract Noninvasive imaging of the brain of animal models demands the detection of increasingly smaller structures by in vivo MRI. The purpose of this work was to elucidate the spatial resolution and structural contrast that can be obtained for studying the brain of C57BL/6J mice by optimized T2-weighted fast spin-echo MRI at 9.4 T. As a prerequisite for high-resolution imaging in vivo, motion artifacts were abolished by combining volatile anesthetics and positive pressure ventilation with a specially designed animal bed for fixation. Multiple substructures in the cortex, olfactory bulb, hippocampus, and cerebellum were resolved at 30 to 40 μm in-plane resolution and 200 to 300 μm section thickness as well as for relatively long echo times of 65 to 82 ms. In particular, the approach resulted in the differentiation of up to five cortical layers. In the olfactory bulb the images unraveled the mitral cell layer which has a thickness of mostly single cells. In the hippocampus at least five substructures could be separated. The molecular layer, Purkinje layer, and granular layer of the cerebellum could be clearly differentiated from the white matter. In conclusion, even without the use of a contrast agent, suitable adjustments of a widely available T2-weighted MRI sequence at high field allow for structural MRI of living mice at near single-cell layer resolution.