The most common MR-based approach to noninvasively measure brain temperature relies on the linear relationship between the 1H MR resonance frequency of tissue water and the tissue's temperature. Herein we provide the most accurate in vivo assessment existing thus far of such a relationship. It was derived by acquiring in vivo MR spectra from a rat brain using a high field (11.74 Tesla [T]) MRI scanner and a single-voxel MR spectroscopy technique based on a LASER pulse sequence. Data were analyzed using three different methods to estimate the 1H resonance frequencies of water and the metabolites NAA, Cho, and Cr, which are used as temperature-independent internal (frequency) references. Standard modeling of frequency-do-main data as composed of resonances characterized by Lorentzian line shapes gave the tightest resonance-frequency versus temperature correlation. An analysis of the uncertainty in temperature estimation has shown that the major limiting factor is an error in estimating the metabolite frequency. For example, for a metabolite resonance linewidth of 8 Hz, signal sampling rate of 2 Hz and SNR of 5, an accuracy of approximately 0.5°C can be achieved at a magnetic field of 3T. For comparison, in the current study conducted at 11.74T, the temperature estimation error was approximately 0.1°C.