ABSTRACTHigh molecular weight glutenin subunits (HMW‐GS) from three hexaploid wheat species (AABBDD, 2n=6x=42, Triticum aestivum L., T. spelta L., and T. compactum L.) were separated and identified by acidic capillary electrophoresis (A‐CE) with phosphate‐glycine buffer (pH 2.5) in uncoated fused‐silica capillaries (50 μm, i.d. × 25.5 cm) at 12.5 kV and 40°C. The rapid separations (<15 min) of HMW‐GS with good repeatability (RSD < 2%) were obtained using a fast capillary rising protocol. All 17 HMW‐GS analyzed could be well separated and their relative migration orders were ranked. In particular, the good quality subunit pair 5+10 could be differentiated from poor quality subunit pair 2+12. In addition, the other three allelic pairs of 13+16, 17+18, and 7+8 subunits that were considered to have positive effects on dough properties, as well as three pairs of novel subunits 13+22*, 13*+19*, and 6.1+22.1 detected from spelt and club wheat, can also be readily separated and identified. An additional protein subunit presented in Chinese bread wheat cultivar Jing 411 and club wheat TRI 4445/75, respectively, was detected by both A‐CE and 2‐D gel electrophoresis (A‐PAGE × SDS‐PAGE), for which further identification is needed.