Microarray-based markers such as Diversity Arrays Technology (DArT) have become the genetic markers of choice for construction of high-density maps, quantitative trait loci (QTL) mapping and genetic diversity analysis based on their efficiency and low cost. More recently, the DArT technology was further developed in combination with high-throughput next-generation sequencing (NGS) technologies to generate the DArTseq platform representing a new sequencing tool of complexity-reduced representations. In this study, we used DArTseq markers to investigate genetic diversity and genome-wide association studies (GWAS) of grain quality traits in rice (Oryza sativa L.). The study was performed using 59 rice genotypes with 525 SNPs derived from DArTseq platform. Population structure analysis revealed only two distinct genetic clusters where genotypes were grouped based on environmental adaptation and pedigree information. Analysis of molecular variance indicated a low degree of differentiation among populations suggesting the need for broadening the genetic base of the current germplasm collection. GWAS revealed 22 significant associations between DArTseq-derived SNP markers and rice grain quality traits in the test genotypes. In general, 2 of the 22 significant associations were in chromosomal regions where the QTLs associated with the given traits had previously been reported, the other 20 significant SNP marker loci were indicative of the likelihood discovery of novel alleles associated with rice grain quality traits. DArTseq-derived SNP markers that include SNP12_100006178, SNP13_3052560 and SNP14_3057360 individually co-localised with two functional gene groups that were associated with QTLs for grain width and grain length to width ratio on chromosome 3, indicating trait dependency or pleiotropic-effect loci. This study demonstrated that DArTseq markers were useful genomic resources for genome-wide association studies of rice grain quality traits to accelerate varietal development and release.