The application of ATR-FTIR spectroscopy for fatty acid content determination provided results statistically similar to GC analysis. The analytical procedure of this method is simple compared to the GC reference method in terms of sample handling and no toxic chemical reagents are needed. Furthermore,the scanning of ATR-FTIR spectra and the analysis time of each sample is less than 5 minutes. This method would be very effective for determination of seed sample fatty acid in breeding programs, when there are large populations. The ATR-FTIR technique using the spectral ranges between 3050-2800 cm and 1800-900 cm with second derivative pretreatment MSC or vector nor malization preprocessing is suitable for the screening of oleic and linoleic acid contents in seed samples. The prediction models can deter mine the concentrations of oleic and linoleic acid with reasonable accuracy. Therefore, the ATR-FTIR technique can be used as an alternative method in plant breeding programs for fatty acid determination in seed samples since it is accurate, rapid and simple.