Cultivar mixtures have the potential to mitigate abiotic stress and stabilize crop yields, but their belowground dynamics remain poorly understood. We evaluated phosphorus (P) uptake by two contrasting spring barley (Hordeum vulgare L.) cultivars (“Anneli” and “Feedway”), grown either in 50:50 mixture or as pure stand. The cultivars were grown in mesocosms under four P fertilization treatments: low‐P, homogeneous high‐P (90 mg P/kg), and localized P hotspots (100 mg P) placed either in the topsoil (5 cm) or subsoil (35 cm). To trace P uptake pathways, the hotspots were labeled with 33P and enclosed in mesh bags allowing only mycorrhizal hyphae (25 μm) or both roots and hyphae (2 mm) to access the hotspot. After 35 days, we measured aboveground biomass, total P content, 33P specific activity, and root biomass, length, diameter, and arbuscular mycorrhiza fungi (AMF) root colonization. In the mixture, reduced P uptake by “Feedway” led to lower overall performance compared to pure stand. Root modifications in the mixture did not enhance biomass or P acquisition, potentially due to decreased AMF colonization. Although different P placements altered P uptake patterns, they did not increase total P uptake. Roots accessed the P hotspots and acquired 33P without notable proliferation in the enriched zones. Our findings underscore the complexity of belowground interactions involving root distribution, competition for P, and AMF, and highlight the need for future research to optimize nutrient acquisition and performance in cultivar mixtures.