Biomarkers of food intake (BFIs) for whole grains (WGs) would enable more precise dietary assessments and help investigate WG's health effects. However, no reports have been published on the biomarkers that reflect the intake of WG wheat in the Chinese diet. In an acute, randomized, crossover intervention study performed on 22 Chinese subjects, WG wheat BFI candidates were screened using an LC-HRMS untargeted metabolomics technique. Screening results indicate that alkylresorcinol (AR) metabolites are WG wheat-specific metabolites. These metabolites were systematically characterized by in vitro metabolism reaction, and the matched high-throughput LC-MS/MS-targeted quantitative method was developed. Time-response plots generated via targeted analysis revealed AR oxidation products (AR-OOH) and their sulfate conjugates (AR-OOH-sul) increased rapidly following the WG wheat consumption, which were identified as novel and short-term WG wheat BFIs. Another attractive biomarker was glucuronidated ARs (AR-glu), which can distinguish between WG and refined grain (RG) groups 24 h after WG wheat ingestion and is considered a promising medium- and long-term biomarker. Subsequently an independent dose-response study was performed for 38 volunteers who consumed different WG wheat amounts (0, 25, 50 or 100 g) for further validation. Identified BFIs perform well in classifying participants into WG wheat consumers or non-consumers, and could capture dose-dependent changes with WG intake increased. Overall, this is the first study to discover and validate WG BFIs in the Chinese population via dietary intervention trials, indicating the potential usefulness for WG wheat intake assessments and dietary compliance monitoring. Future work will examine their use in large-scale free-living populations.