We have developed a method for measuring the temperature of micro-regions in aqueous solutions using near-infrared spectroscopy that enables us to measure the temperature of biological cells, tissues, and biochemical solutions in vitro. The measurement principle is based on the fact that the peak wavelength of the water absorption band with its center near 1450 nm shifts with changes in temperature. The measurement system, which consists of a biological microscope and two spectrophotometers, can measure respective absorbance spectra for two areas that are each 80 microm in diameter. We formed the temperature distribution in a 500-microm thick water film by heating an immersed Nichrome wire and measured the temperature difference between the two areas. The results of the measurement were compared to a calculated temperature distribution.