Rubber-based seismic isolation has been demonstrated to be one of the most effective measures to protect structural elements from damage during earthquakes and a viable option to retrofit existing structures with poor seismic detailing. The main constituent of these isolation units is rubber, a material that is subject to stiffening when exposed to low air temperatures. In the case of isolated highway bridges, thermal stiffening might reduce the efficiency of isolators, transferring higher forces to the substructure. Assessment of the seismic response of retrofitted structures using rubber isolators in cold regions is thus necessary. Accordingly, in this study, the effect of low temperatures on the seismic performance of a highway bridge retrofitted with natural rubber (NR) isolators is quantified using a probabilistic framework based on fragility surfaces. From the component- and system-level surfaces, it is revealed that the effects of cold temperatures on highway bridges retrofitted with elastomeric isolators may be negligible, depending on the configuration of lateral restraining structures. However, when isolators are able to perform their function without impediment, their thermal stiffening might be significantly detrimental to the bridge’s substructure, mainly affecting bent columns.