The increasing number of mouse models of human disease used in biomedical research applications has led to an enhanced interest in non‐invasive imaging of mice, e.g. using MRI for phenotyping. However, MRI of small rodents puts high demands on the sensitivity of data acquisition. This requirement can be addressed by using cryogenic radio‐frequency (RF) detection devices. The aim of this work was to investigate the in vivo performance of a 400 MHz cryogenic transmit/receive RF probe (CryoProbe) designed for MRI of the mouse brain. To characterize this novel probe, MR data sets were acquired with both the CryoProbe and a matched conventional receive‐only surface coil operating at room temperature (RT) using conventional acquisition protocols (gradient and spin echo) with identical parameter settings. Quantitative comparisons in phantom and in vivo experiments revealed gains in the signal‐to‐noise ratio (SNR) of 2.4 and 2.5, respectively. The increased sensitivity of the CryoProbe was invested to enhance the image quality of high resolution structural images acquired in scan times compatible with routine operation (< 45 min). In high resolution (30 × 30 × 300 µm³) structural images of the mouse cerebellum, anatomical details such as Purkinje cell and molecular layers could be identified. Similarly, isotropic (60 × 60 × 60 µm³) imaging of mouse cortical and subcortical areas revealed anatomical structures smaller than 100 µm. Finally, 3D MR angiography (52 × 80 × 80 µm³) of the brain vasculature enabled the detailed reconstruction of intracranial vessels (anterior and middle cerebral artery). In conclusion, this low temperature detection device represents an attractive option to increase the performance of small animal MR systems operating at 9.4 Tesla. Copyright © 2009 John Wiley & Sons, Ltd.