The chemical method for the determination of the resistant starch (RS) content in grains is time-consuming and labor intensive. Near-infrared (NIR) and attenuated total reflectance mid-infrared (ATR-MIR) spectroscopy are rapid and nondestructive analytical techniques for determining grain quality. This study was the first report to establish and compare these two spectroscopic techniques for determining the RS content in wheat grains. Calibration models with four preprocessing techniques based on the partial least squares (PLS) algorithm were built. In the NIR technique, the mean normalization + Savitzky-Golay smoothing (MN + SGS) preprocessing technique had a higher coefficient of determination (R _c ² = 0.672; R _p ² = 0.552) and a relative lower root mean square error value (RMSEC = 0.385; RMSEP = 0.459). In the ATR-MIR technique, the baseline preprocessing method exhibited a better performance regarding to the values of coefficient of determination (R _c ² = 0.927; R _p ² = 0.828) and mean square error value (RMSEC = 0.153; RMSEP = 0.284). The validation of the developed best NIR and ATR-MIR calibration models showed that the ATR-MIR best calibration model has a better RS prediction ability than the NIR best calibration model. Two high grain RS content wheat mutants were screened out by the ATR-MIR best calibration model from the wheat mutant library. There was no significant difference between the predicted values and chemical measured values in the two high RS content mutants. It proved that the ATR-MIR model can be a perfect substitute in RS measuring. All the results indicated that the ATR-MIR spectroscopy with improved screening efficiency can be used as a fast, rapid, and nondestructive method in high grain RS content wheat breeding.

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