Tri-step infrared spectroscopy method of Fourier transform infrared spectroscopy, second derivative infrared spectroscopy and two-dimensional correlation infrared spectroscopy was firstly used to discriminate six species of mushrooms belonging to the genus Lactarius and Russula. The absorption bands of the original spectrum were very similar, which were composed by protein and polysaccharides, but tiny differences were observed at the position, shape and absorption intensities of peaks. Second derivative infrared spectroscopy technology was applied to study 6 species of the samples, there were obvious differences in the range of 1 800～1 400 and 1 200～800 cm-1. Two-dimensional correlation infrared spectroscopy can improve the resolution of spectra. Therefore two-dimensional correlation infrared spectroscopy was used to study 6 kinds of mushrooms. The results showed that there are three auto-peaks in the Lactarius, four in the Russula and significant differences in the number, intensity of auto-peaks and cross peaks were observed in the range of 1 690~1 420 cm-1. In addition, the peaks quantity, position, intensity of auto-peaks and cross peaks were different in the range of 1 110~920 cm-1. It demonstrates that tri-step infrared spectroscopy technology of Fourier transform infrared spectroscopy, second derivative infrared spectroscopy and two-dimensional correlation infrared spectroscopy is a rapid and effective method for discriminating Lactarius and Russula.