In this paper, the crop growth model DAISY for spring barley (cultivar “Tolar“) was calibrated and subsequently validated in three different soil-climate locations in the Czech Republic – Lednice (48°48'51'' N, 16°48'46'' E, altitude 180 m), Věrovany (49°27'39'' N, 17°17'42'' E, altitude 210 m) and Domanínek (49°31'42'' N, 16°14'13'' E, altitude 560 m). The calibration and validation were based on data from a multi-year field experiment from the Central Institute for Supervising and Testing in Agriculture and from a two-year field experiment in Domanínek (2011 and 2012) that was conducted by the Institute of Agrosystems and Bioclimatology in cooperation with the Global Change Research Centre AS CR. The calibration for Lednice, Věrovany and Domanínek was performed using 4 growth seasons from each station, the subsequent validation for Lednice and Věrovany was performed based on 3 growth seasons from each station, and that for Domanínek was based on 6 growth seasons. The value of the RMSE (root mean square error) statistic for flowering was 2 days for calibration and 4 days for validation on average; for maturity, the RMSE was 11 days for both calibration and validation. The average RMSE for the yields was 0.9 t·ha−1 for calibration and 1.6 t·ha−1 for validation. According to the statistical index MBE (mean bias error) for the flowering phenological phase, the crop growth model DAISY showed a delay of 2 days in both calibration and validation. There was also delay of 6 days in calibration and of 8 days in validation for maturity. According to the MBE, the crop growth model DAISY underestimates the yield by 0.2 t·ha−1 for calibration and underestimates the yield by 0.4 t·ha−1 for validation.