In this study, temperatures of multiple gas slabs are measured from a single absorption line using a tunable diode laser absorption spectroscopy technique. While typical laser absorption spectroscopy techniques require two or more absorption lines to determine the gas temperatures, however, this study presents the temperature determination method using an absorption line and its conditions for successful application. To generate two air slabs along with a single laser path, a gas heating apparatus composed of two chambers is used. The air is used as a test gas and a tunable diode laser absorption spectroscopy system is set up to detect an absorption line of the oxygen molecule in the air. Using the spectroscopic characteristics that the line shape and absorption strength are functions of gas properties and beam path length, temperature values of multiple gas slabs are experimentally determined by matching a measured spectral absorbance with a calculated one with known pressure and beam path length values. The determined temperatures are compared with temperature values set using thermocouples. It is observed that the temperatures of the gas slabs can be successfully measured when the pressures of the gas slabs are considerably different. The effect of gas properties and beam path length on the temperature determination is discussed. Measurement uncertainty due to random noise in the signal is investigated through a simulation approach.