Assessment of genetic diversity and molecular characterization among elite rice varieties of Bangladesh is very important for germplasm management, varietal identification, and DNA fingerprinting. Thirty-four microsatellite markers were studied across 21 types of rice to characterize and discriminate among different varieties. The number of alleles per locus ranged from 2 to 11, with an average of 4.18 alleles across 34 loci. A total of 57 rare alleles were detected at 24 loci, whereas 42 unique alleles were detected at 20 loci. The results revealed that 14 rice varieties produced unique alleles that could be used for identification, molecular characterization, and DNA fingerprinting of these varieties. Polymorphic information content (PIC) values ranged from 0.157 to 0.838, with an average of 0.488, which revealed that much variation was present among the studied varieties. The PIC values revealed that RM401 might be the best marker for identification and diversity estimation of rice varieties, followed by RM566, RM3428, RM463, and RM8094 markers. The UPGMA cluster dendrogram created in this study identified five clusters with a similarity coefficient of 0.50. The SSR polymorphism and diversity could likely be attributed to pedigree. In this study, eight SSR markers (RM10713, RM279, RM424, RM6266, RM1155, RM289, RM20224, and RM5371) were identified that produced specific alleles only in the aromatic rice varieties and were useful for varietal identification and DNA fingerprinting of these varieties. The findings of this study should be useful for varietal identification and could help in background selection in backcross breeding programs.