This study aims to evaluate the effect of various background reference regions on spinal 18F-FET PET imaging, with a focus on distinguishing between spinal tumors and myelitis. To enhance diagnostic accuracy, we investigated the pons and several other spinal cord area as potential references, given the challenges in interpreting spinal PET results.A retrospective analysis was conducted on 30 patients, 15 with cervical myelitis and 15 with cervical tumors, who underwent O-(2-[18F]-fluoroethyl)-L-tyrosine (FET) PET/MR imaging. The stability of uptake across four regions, including the pons, C2, C2-C7, and T1-T3, was compared. The standardized uptake value ratio (SUVR) was then evaluated using various background regions, and their effectiveness in differentiating between spinal tumors and myelitis was compared. Additionally, we correlated the SUVR values derived from these regions with the Ki-67 proliferation index in tumor patients. The study found no significant difference in SUVmax (U = 110, p = 0.93) and SUVmean (U = 89, p = 0.35) values at lesion sites between myelitis and tumor patients. The pons had the highest average uptake (p < 0.001) compared to the other three regions. However, its coefficient of variation (CV) was significantly lower than that of the C2-C7 (p < 0.0001) and T1-T3 segments (p < 0.05). The SUVRmax values, calculated using the regions of pons, C2-C7 and T1-T3, were found to significantly differentiate between tumors and myelitis (p < 0.05). However, only the pons-based SUVRmean was able to significantly distinguish between the two groups (p < 0.05). Additionally, the pons-based SUVRmax (r = 0.63, p = 0.013) and SUVRmean (r = 0.67, p = 0.007) demonstrated a significant positive correlation with the Ki-67 index.This study suggests that the pons may be considered a suitable reference region for spinal 18F-FET PET imaging, which can improve the differentiation between spinal tumors and myelitis. The significant correlation between pons-based SUVR values and the Ki-67 index further highlights the potential of this approach in assessing tumor cell proliferation.© 2024. The Author(s).