The transition towards zero carbon emissions in power generation hinges on the integration of efficient electrical energy storage systems, with lithium-ion batteries (LIBs) positioned as a pivotal technology. While generally safe, deviations in their operational guidelines due to manufacturing defects or misuse can lead to critical safety concerns, notably thermal runaway (TR) events. Internal short circuits (ISCs) are primary initiators of TR within LIBs. For abuse testing, ISCs are often triggered by nail penetration. This study explores the morphological changes and mechanisms underlying ISC-induced TR in LIBs using operando synchrotron X-ray computed tomography (SXCT) at subzero temperatures. A novel cryogenic setup was developed to control a stepwise temperature increase in the damaged sample while monitoring electrochemical characteristics and simultaneously enabling acquisition of high-resolution SXCT images. The findings reveal that conducting nail penetration at minus 80 °C prevents immediate TR, enabling detailed analysis of subsequent structural and electrochemical behavior during controlled thawing. Thus, the initiation of TR processes at localized ISC sites has been observed, evidenced by voltage fluctuations and morphological changes, such as cathode material cracking and decomposition. These results underscore the importance of temperature control in mitigating TR risks and provide critical insights into the internal dynamics of LIBs under abusive conditions. The developed cryogenic SXCT methodology offers a powerful tool for non-destructive, high-resolution investigation of battery failure mechanisms, contributing to the enhancement of LIB safety.