PurposeThe presence of orientation-dependent susceptibility artifacts in magnetic resonance chemical shift thermometry (CST) can confound accurate temperature calculations. Here, we quantify the effect of white matter (WM) tract orientation on CST due to tissue-specific susceptibility. MethodsTwenty-nine healthy volunteers (27 ± 4 years old) were scanned on a 3 T MR scanner with a 32-channel head coil. Diffusion tensor imaging (DTI), T1-weighted imaging, and single voxel spectroscopy (SVS) for CST were acquired. Participants were then asked to rotate their head ∼3–5° (yaw or roll) to alter the orientation of WM tracts relative to the external magnetic field. After head rotation, a second SVS scan and T1-weighted imaging were acquired. The WM-fraction-normalized DTI principal eigenvector (V1) images were used to calculate the length of the x-y component of V1, which was used as a surrogate for WM tracts perpendicular to B0. A linear regression model was used to determine the relationship between the perpendicular WM tracts and brain temperature. ResultsSignificant temperature differences between post- and pre-head rotation scans were observed for brain (−0.72 °C ± 1.36 °C, p = 0.01) but not body (0.012 °C ± 0.07 °C, p = 0.37) temperatures. The difference in brain temperature was positively associated with the corresponding change in perpendicular WM tracts after head rotation (R2 = 0.26, p = 0.005). ConclusionOur results indicate WM tract orientation affects temperature calculations, suggesting artifacts from orientation-dependent susceptibility may be present in CST.