Background: Weeds reduce wheat yields in dryland farming systems. Herbicides such as metribuzin are commonly used to control weeds. However, wheat has a narrow safety margin against metribuzin. Standing crops such as wheat with weeds in the same field can also be killed by the same dose of metribuzin. Therefore, it is important to identify metribuzin resistance genes and understand the resistance mechanism in wheat for sustainable crop production. A previous study identified a significant metribuzin resistance wheat QTL, Qsns.uwa.4 A.2, explaining 69% of the phenotypic variance for metribuzin resistance. Results: Two NIL pairs with the most contrasting performance in the metribuzin treatment and different in genetic backgrounds were compared using RNA sequence analysis, identifying nine candidate genes underlying Qsns.uwa.4 A.2 responsible for metribuzin resistance. Quantitative RT-qPCR further validated the candidate genes, with TraesCS4A03G1099000 (nitrate excretion transporter), TraesCS4A03G1181300 (aspartyl protease), and TraesCS4A03G0741300 (glycine-rich proteins) identified as key factors for metribuzin resistance. Conclusion: Identified markers and key candidate genes can be used for selecting metribuzin resistance in wheat.