Abstract: It is important to find suitable alternatives for medicinal plants due to the gradual decline of wild resources. Tissue culture exhibits significant advantages in achieving medicinal plant substitutes. However, few studies have reported on the application of Fourier transform infrared (FTIR) spectroscopy to select appropriate material. In this research, FTIR combined with partial least squares discriminant analysis (PLS-DA) was used to compare calli (fleshy part, stem and leaf), proliferation plantlets (fleshy part, stem and leaf) and regenerated plantlets (root, stem and leaf) of Gentiana rigescens Franch. ex Hemsl. formed by tissue culture. Results showed that:(1) FTIR spectra of the fleshy parts and roots of G. rigescens samples were alike, as were the stems and leaves; (2) Second derivative spectra showed clear differences among the samples. Around the main characteristic absorption peak (1612 cm^-1) of gentiopicroside, the intensities of absorption peak was, in turn, the regenerated plantlet leaf, proliferation plantlet leaf and regenerated plantlet stem, proliferation plantlet stem. However, the stem and fleshy part of the callus, fleshy part of the proliferation plantlet and the root of the regenerated plantlet had no spectral peaks in this position; (3) Results of PLS-DA demonstrated that samples of the same part and at the same tissue culture stage could be grouped together. The fleshy parts and roots differed from the stems and leaves of the samples. Thus, the chemical constituents and content of the stems and leaves of G. rigescens could be differentiated from the fleshy parts and roots, with the fleshy parts similar to the roots and the stems similar to the leaves. Second derivative spectra showed that material formed by tissue culture could be a viable alternative to the original plants for medical use. In addition, regenerated plantlet leaves exhibited great potential for exploitation based on gentiopicroside, and may replace wild G. rigescens to relieve resource scarcity. Our study showed that FTIR can be used as a simple and effective method for the preliminary assessment of the substitution potential and utilization of different parts of medicinal plants during different stages in tissue culture.