Rice is an important human staple food for over half of the world’s population. Amylose content (AC), gelatinization temperature (GT), grain protein content (GPC), percentage grain chalkiness (PGC), and mineral content are important parameters for evaluating rice quality, which attracts customers and breeders. Only limited genes or QTLs (OsAAP6, OsGluA2, OsASN1, Chalk5, OsHMA3, etc.) are reported regulating rice GPC, PGC, and mineral content due to the lack of genetic knowledge and molecular markers. To dissect the genetic architecture of rice grain quality regulation, genome wide association studies (GWAS) were performed using two populations (USDA-mini core collection and a panel of 662 rice accessions from the 3K Rice Genomes Project). A total of 28, 11, 4, 3, 40, 3, 4, 3, and 10 QTLs were identified associated with Cd, Co, Cu, K, Mo, Ni, Rb, Sr, and Zn under flooded environment, while, 23, 7, 7, 7, and 3 QTLs were detected to be associated with Cd, Fe, Mo, Ni, and Zn under unflooded environment, respectively. Moreover, 6, 5, and 2 significant QTLs were tightly associated with kernel length, kernel rate, kernel width, respectively. Furthermore, 44, 7, 27, and 20 QTLs were identified associated with AC, GT, GPC, and PGC, respectively. Overall, 53 (~ 20.08%) of the 264 QTLs were coinciding with previously reported QTLs/genes, and 211 (~ 79.92%) were novel QTLs. A candidate gene, OsPCAT (putative cationic transporter), associated with GPC in the dry season was selected for further analysis. The OsPCAT gene belongs to the amino acid transporters (AATs) family with nine closely related members reported in Oryza Sativa. The classification and evolution of the CAT family (a subgroup of AATs family) using 61 species were studied. The over-expression lines (OsPCAT-OX) and CRISPR-Cas9 knock-out lines (OsPCAT-KO) were developed to study the function of OsPCAT gene. The preliminary results showed the GPC in OsPCAT-OX lines was increased and OsPCAT-KO lines were decreased compared to WT. Overall, a large number of new and reported QTLs associated with rice grain quality have been identified. This work lays the foundation for marker development in breeding and further investigation on rice grain quality regulation.