The aims of this study were to compare the responses of four winter wheat cultivars to nitrogen fertilization with vegetation indices calculated using spectral reflection (GreenSeeker hand-held sensor) and to estimate in-season yield (INSEY) using the vegetation indices. The field experiment was conducted at Transitional Zone Agricultural Research Institute of Eskisehir province, Turkey in 2007-2008, 2008-2009 and 2009-2010 growing seasons. The experimental layout was a 2factor factorial in the randomized complete block design. Nitrogen rates were 0, 40, 80, 120, 160 and 200 kg N ha^-1. Vegetation Index (NDVI) was obtained at growth stages of Zadoks 24 (tillering stage), Zadoks stage 30 (stem elongation), Zadoks stage 31 (the first node is visible) and Zadoks stage 32 (the second node is visible). The results revealed that Zadoks stage 30 was the most realistic reading time. NDVI had the advantage of providing information on biomass, in addition to nitrogen nutrition status of crops, enabling in-season yield estimation possible. Therefore, NDVI based calibration equations were preferred for testing in the fields of actual farmers for the last year of study. A comparison of the system with traditional farmer applications indicated that yield estimation obtained by the new system was quite similar yields with 13.2 kg ha^-1 less N in the spring (ZD 3.0), showing its economically promising value.