This study applies three methods, Gene Expression Programming (GEP), M5 tree (M5T) model and optimized Artificial Neural Network by Genetic Algorithm (ANN-GA) for estimation of reference evapotranspiration in Ahvaz and Dezful in the southwest of Iran. Comparison between results of the FAO Penman-Monteith (FPM) method and the mentioned three methods shows that ANN-GA with the Levenberg-Marquardt training method is the best method and the M5T model is the second appropriate method for estimation of reference evapotranspiration. In Ahvaz, R2 and RMSE of ANN-GA method are 0.996, 0.184 mm/day. For M5T method, these values are 0.997 and 0259 mm/day, and for GEP method, they are 0.979 and 0.521 mm/day. In Dezful, R2 and RMSE of ANN-GA method are 0.994, 0.235 mm/day. For M5T method, these values are 0.992 and 0265 mm/day, and for GEP method, they are 0.963 and 0.544 mm/day. In addition, sensitivity analysis shows that the maximum temperature is the most effective parameter, and the wind speed is second effective parameter. In Dezful, the effect of the maximum temperature is more than those of Ahvaz but the effect of wind speed is less than those of Ahvaz. Because Ahvaz is more flatter than Dezful (the movement of wind in Ahvaz is freer than those of Dezful). The third effective meteorological parameter is the average relative humidity in Ahvaz and the sunny hours in Dezful. The reason for this subject is the less distant of Ahvaz from the Persian Gulf (it is source of moisture).