Rotational temperatures in the Mesosphere‐Lower Thermosphere region are estimated by utilizing the OH(6,2) Meinel band nightglow data obtained with an Ebert‐Fastie spectrometer (EFS) operated at Arecibo Observatory (AO), Puerto Rico (18.35°N, 66.75°W) during February‐April 2005. To validate the estimated rotational temperatures, a comparison with temperatures obtained from a co‐located Potassium Temperature Lidar (K‐Lidar) and overhead passes of the Sounding of the Atmosphere by Broadband Emission Radiometry (SABER) instrument onboard NASA's Thermosphere Ionosphere Mesosphere Energetics and Dynamics (TIMED) satellite is performed. Two types of weighting functions are applied to the K‐Lidar temperature profiles to compare them with EFS temperatures. The first type has a fixed peak altitude and a fixed full width at half maximum (FWHM) for the whole night. In the second type, the peak altitude and FWHM vary with the local time. SABER measurements are utilized to estimate the OH(6,2) band peak altitudes and FWHMs as a function of local time and considerable temporal variations are observed in both the parameters. The average temperature differences between the EFS and K‐Lidar obtained with both types of weighting functions are comparable with previously published results from different latitude‐longitude sectors. We found that the temperature comparison improves when the time‐varying weighting functions are considered. Comparison between EFS, K‐Lidar, and SABER temperatures reveal that on average, SABER temperatures are lower than the other two instruments and K‐Lidar temperatures compare better with SABER in comparison to EFS. Such a detailed study using the AO EFS data has not been carried out previously.