The purpose of this study was to determine the effect of sixty years of contrasting fertilization treatments on the roots of winter wheat (Triticum aestivum L.) at sites with different soil and climate conditions. The depth and length density distribution of the wheat roots were determined between 2014 and 2016 in a crop rotation experiment established in 1955 at three sites: Lukavec, Čáslav, and Ivanovice (Czech Republic). Three fertilization treatments were examined: Zero fertilization (N0), organic (ORG) fertilization, and mineral (MIN) fertilization. The fertilization, site, and year all had a significant effect on the total root length (TRL). The average TRL per square meter reached 30.2, 37.0, and 46.1 km with the N0, ORG, and MIN treatments at Lukavec, respectively, which was the site with the lightest soil and the coldest climate. At Čáslav and Ivanovice (warmer sites with silt and loamy soils), the average TRL per square meter reached 41.2, 42.4, and 47.7 km at Čáslav and 49.2, 55.3, and 62.9 km at Ivanovice with the N0, MIN, and ORG treatments, respectively. The effect of fertilization on the effective root depth (EfRD), the depth at which the root length density dropped below 2.0 cm cm−3, was significant, while the maximum root depth (RMD) was only marginally affected. With the sites and years averaged, the MIN-treated plants showed a greater EfRD (102.2 cm) in comparison to the N0 (81.8 cm) and ORG (93.5 cm) treatments. The N0 treatment showed no signs of an adaptive reaction to the root system, with potential improvement for nutrient acquisition, while optimal fertilization contributed to the potential for resource depletion from the soil profile.