The suppression level of internal stray radiation is a key indicator to evaluate infrared imaging systems. Being related to ambient temperature, the internal stray radiation must be measured at multiple ambient temperatures, and the measurement has such disadvantages as high cost, long duration and high demand for experimental setups. To solve these problems, the effect of ambient temperature on internal stray radiation is studied by building a multi-integral time calibration model, and a method is proposed to measure internal stray radiation of cooled infrared imaging systems using ambient temperature. In this method, the influence of internal factors of the detector on the system output is obtained by calibrating the cooled infrared detector. Combining the calibration results of the infrared imaging system under a certain ambient temperature, the quantitative relation between internal stray radiation and ambient temperature is resolved. Then the internal stray radiation can be calculated at arbitrary integration times and ambient temperatures. The effectiveness of the proposed method is verified by radiometric calibration experiments. Experimental results indicate that, with the proposed method, high-precision measurement of internal stray radiation in cooled infrared imaging systems can be achieved.