•A method to image tissue frozen at autopsy, without causing tissue degradation during MRI, is described here.•First, RNA degradation rate was measured at various temperatures to determine a range of optimal conditions for MRI.•A refrigeration system was set up inside the MRI to allow reliable temperature maintenance during imaging.•We designed biosafe tissue holder for MRI, and transport system to allow imaging of human autopsy tissue.•MRI of tissue-blocks using an inversion-prepared sequence shows anatomical details and pathological regions. Magnetic Resonance Imaging (MRI) can provide the location and signal characteristics of pathological regions within a postmortem tissue block, thereby improving the efficiency of histopathological studies. However, such postmortem-MRI guided histopathological studies have so far only been performed on fixed samples as imaging tissue frozen at the time of extraction, while preserving its integrity, is significantly more challenging. Here we describe the development of cold-postmortem-MRI, which can preserve tissue integrity and help target techniques such as transcriptomics. As a first step, RNA integrity number (RIN) was used to determine the rate of tissue biomolecular degradation in mouse brains placed at various temperatures between -20 °C and +20 °C for up to 24 h. Then, human tissue frozen at the time of autopsy was immersed in 2-methylbutane, sealed in a bio-safe tissue chamber, and cooled in the MRI using a recirculating chiller to determine MRI signal characteristics. The optimal imaging temperature, which did not show significant RIN deterioration for over 12 h, at the same time giving robust MRI signal and contrast between brain tissue types was deemed to be −7 °C. Finally, MRI was performed on human tissue blocks at this optimal imaging temperatures using a magnetization-prepared rapid gradient echo (MPRAGE, isotropic resolution between 0.3–0.4 mm) revealing good gray-white matter contrast and revealing subpial, subcortical, and deep white matter lesions. RINs measured before and after imaging revealed no significant changes (n = 3, p = 0.18, paired t-test). In addition to improving efficiency of downstream processes, imaging tissue at sub-zero temperatures may also improve our understanding of compartment specificity of MRI signal.