Variety mixtures combining crop varieties with different root system properties have the potential to improve soil exploration through belowground niche complementarity, which can improve soil resource acquisition and crop productivity. However, there is a lack of appropriate methods to distinguish and quantify roots of different varieties, which limits our ability to elucidate belowground processes that underpin soil exploration and resource uptake by plants in variety mixtures. In the present study, we developed a method to quantify root biomass and distribution patterns of different barley varieties grown together in mixtures using DNA extraction and quantitative PCR with variety-specific genetic markers. Two field experiments, one in Sweden and one in Denmark, were conducted that included two barley varieties grown either alone in pure stands or together in the same plot. The genetic markers were highly variety-specific, enabling accurate detection of the roots of each individual variety in the mixture. We found that the contribution of varieties to total root biomass in the mixture differed between the two locations, indicating the effects of the environment on root distribution patterns in variety mixtures. The method presented here opens new possibilities for rapid quantification of root biomass and can provide new insights into belowground processes underpinning the functioning of mixed variety systems. Ultimately, such understanding is needed to assess the potential to adopt mixed variety systems in practical agriculture.