Wheat is the most important staple crop in Afghanistan and accounts for the main part of cereal production. However, wheat production has been unstable during the last decades and the country depends on seed imports. Wheat research in Afghanistan has emphasized releases of new, high-yielding and disease resistant varieties but rates of adoption of improved varieties are uncertain. We applied DNA fingerprinting to assess wheat varieties grown in farmers’ fields in four Afghan provinces. Of 560 samples collected from farmers’ fields during the 2015-16 cropping season, 74% were identified as varieties released after 2000, which was more than the number reported by farmers and indicates the general prevalence of use of improved varieties, albeit unknowingly. At the same time, we found that local varieties and landraces have been replaced and were grown by 4% fewer farmers than previously reported. In 309 cases (59%), farmers correctly identified the variety they were growing, while in 219 cases (42%) farmers did not. We also established a reference library of released varieties, elite breeding lines, and Afghan landraces, which confirms the greater genetic diversity of the landraces and their potential importance as a genetic resource. Our study is the first in wheat to apply DNA fingerprinting at scale for an accurate assessment of wheat varietal adoption and our findings point up the importance of DNA fingerprinting for accuracy in varietal adoption studies.