Amylose content (AC) is one of the key traits related to rice grain quality. It decided the hard or soft grain rice. Consumers generally prefer cooked rice with a soft and sticky texture. AC is a single-gene trait and is controlled by the Waxy gene located on chromosome 6. Genetic studies related to AC in medium-grain rice varieties underpin the identification of genes and linked molecular markers that few previous studies mentioned. The study’s purpose is to investigate the genetic characteristics of AC in medium-grain rice on 12 chromosomes. A rice diversity panel of 114 genotypes was applied to evaluate AC and its correlation with the agronomic and quality characteristics. Genome-Wide Association Study (GWAS) was utilized to identify significant SNPs. The results showed that the AC of varieties had a large range from 10.83 to 30.12%, in which, the group of varieties with low AC (less than 20%) accounted for 21.9%. AC was strongly correlated with gel consistency (GC) but not with gelatinization temperature (GT) and agronomic traits. The GWAS analysis of AC identified 9 significant SNPs on chromosomes 1, 2, 4, 8, 9, 11 and 12. The frequency of alleles widely ranged from 0.94 to 56.60%. On chromosome 6, 74 significant SNPs had allele frequencies ranging from 3.8 to 67.0%. In which, two SNPs fd7 (1764762 bp) and fd8 (176707 bp) are located in the Waxy gene region. These findings provided the basic genetic foundation for high-quality medium-grain rice breeding programs.