The R fluorescence lines of rubies that contain 0.022, 0.068, 0.211, 0.279, 0.556, 1.221, and 1.676 wt% of Cr2O3 were measured at temperatures of 100–600 K and at atmospheric pressure. The R1 line wavenumbers of all of the ruby samples shifted linearly as the temperature increased from 298 to 600 K at atmospheric pressure, and the temperature dependence increased from −0.157 ± 0.001 cm−1/K to −0.149 ± 0.001 cm−1/K as the Cr2O3 content in the rubies increased from 0.022 to 1.676 wt%, which suggests a significant dependence on Cr3+ concentration. At room temperature and atmospheric pressure, the full-width at half maximum (FWHM) of the peak height of the R lines also appears to be linearly related to the Cr3+ concentration. The relative intensity ratios of the R2 to R1 lines (I2/I1) of ruby samples with different Cr3+ concentrations show several non-linear variations with temperature from 100 to 600 K, and the maximum values, (I2/I1)max, occur near room temperature. The effect of Cr3+ doping on the temperature dependence of the R line wavenumbers should be considered when rubies are used to calibrate the pressure or temperature in high-pressure and high-temperature experiments.