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High-amylose maize starch has great potential for widespread industrial use due to its ability to form strong gels and film and in the food processing field, thus serving as a resistant starch source. However, there is still a substantial shortage of high-amylose maize due to the limitation of natural germplasm resources, although the well-known amylose extender (ae) gene mutants have been found to produce high-amylose maize lines since 1948. In this context, high-amylose maize lines (13 inbreds and 18 hybrids) originating from a natural amylose mutant in our testing field were utilized to study the correlation between amylose content (AC) and phenotypic traits (kel morphology and endosperm glossiness), grain filling characteristics, gene expression, and amylose inheritance. Our results showed that AC was negatively correlated with total starch content but was not correlated with grain phenotypes, such as kel fullness, kel morphology and endosperm glossiness. Maize lines with higher amylose had a greater grain filling rate than that of the control (B73) during the first 20 days after pollination (DAP). Both starch debranching enzyme (DBE) groups and starch branching enzyme IIb (SBEIIb) groups showed a greater abundance in the control (B73) than in the high-amylose maize lines. Male parents directly predicted AC of F1, which was moderately positively correlated with the F2 generation.