Fourier transform infrared spectroscopy (FTIR) was used to study three group tobacco leaves of brown spot, angular spot and weather speck, with each group being composed of three samples, namely, leaf spots, near-spot and normal tobacco leaves. The results indicate that the absorption ratio A1 631/A1 025 of the three group tobacco leaves showed the same change tendency, with the normal tobacco leaves＜the near-spot leaves＜the leaf spots. For a more objective and comprehensive analysis, the original and second-derivative spectra were selected for distance analysis in the whole region. The results show that the Pearson correlation coefficient of the near-spot leaves and normal leaves is greater than the corresponding coefficient of leaf spots and normal leaves, which suggest that the near-spot leaves and normal leaves have a closer relationship compared with the leaf spots and normal leaves. The ratios of the A1 631/A1 025 and Pearson correlation coefficients show that the chemical composition of the near-spot leaves changed gradually, that is, the near-spot leaves were in a transient state between normal and disease leaves. In conclusion, FTIR spectroscopy is a promising technique for diagnosing tobacco disease in the incubation period.