The development of high amylose corn (Zea maysL.) in the public sector has been hindered by the lack of high amylose germplasm available to breeders and difficulties in accurate and efficient amylose content measurement. GEMS‐0067 is the first public germplasm that possesses recessive amylose‐extender(ae) gene and a modifier gene or genes that raises amylose level to over 70%. Several lines of evidences suggested that SbeI is one of the major modifier genes for high amylose corn, and our work for the first time showed that SbeI explained 49% of the total amylose variance in the amylose extenderbackground. We developed and validated functional codominant molecular markers of SbeI and SbeIIb for GEMS‐0067 that were able to distinguish AeAe, Aeae, and aeaegenotypes. These simple, sequence‐specific, polymerase chain reaction‐based, low‐cost markers amenable to larger numbers of plant samples make them potentially viable for marker‐assisted selection in practical high amylose corn breeding programs. GEMS‐0067 was of mixed heterotic derivation. Our result clearly showed that GEMS‐0067 was genetically more similar to Mo17 and was clustered into non‐Stiff Stalk heterotic group based on 25 simple sequence repeat profiles. The information provided here would facilitate the development of high amylose corn.