Purpose: This study evaluated the extent of damage due to hypothermia in the mature and immature brain. Methods: Hippocampal tissue cultures at 7 and 14 days in vitro (DIV) were used to represent the immature and mature brain, respectively. The cultures were exposed at $25^{circ}C$ for 0, 10, 30, and 60 minutes (n=30 in each subgroup). Propidium iodide fluorescent images were captured 24 and 48 hours after hypothermic injury. Damaged areas of the cornu ammonis 1 (CA1), CA3, and dentate gyrus (DG) were measured using image analysis. Results: At 7 DIV, the tissues exposed to cold injury for 60 minutes showed increased damage in CA1 (P<0.001) and CA3 (P=0.005) compared to the control group at 48 hours. Increased damage to DG was observed at 24 (P=0.008) and 48 hours (P=0.011). The 14 DIV tissues did not demonstrate any significant differences compared with the control group, except for the tissues exposed for 30 minutes in which DG showed less damage at 48 hours than the control group (P=0.048). In tissues at 7 DIV, CA1 (P=0.040) and DG (P=0.013) showed differences in the duration of cold exposure. Conclusion: The immature brain is more vulnerable to hypothermic injury than the mature brain.