Exploring the diversity at the molecular level is one of the prerequisites in modern plant breeding to address the world food security challenges. Malaysian rice cultivars have been characterized using single sequence repeat (SSR) markers to assess the molecular diversity. In this study, 21 Malaysian rice cultivars were characterized using primers of 27 microsatellite loci to investigate the molecular diversity. The extracted DNA and PCR-based methods were used to amplify the potential microsatellite loci. According to derived results, 61 alleles were amplified including 27 rare and 15 unique alleles and also 88.89% of all studied microsatellite loci were polymorphic. Moreover, di-nucleotide repeat motifs were shown to be more frequent (49.28%) than tri-nucleotide repeat motifs (48.28%). Polymorphism information content (PIC) values were ranged from 0.090 (RM411) to 0.940 (RM105) with an average of 0.531. Furthermore, the UPGMA derived dendrogram grouped all varieties into three major clusters. A high proportion of polymorphism (88.89%) could be due to a relatively high level of genetic diversity among studied cultivars. Chromosome 4 with the highest mean PIC value (0.917) and chromosome 7 (0.607) are very valuable for gene mapping, marker-assisted selection (MAS) amplification and rice breeding programs among Malaysian rice cultivars.