A new rice variety takes about 12 years to release, the number of available distinct morphological features is very small, due to the continual pressure of releasing new varieties. The objectives of this study were: to estimate the genetic variation of Egyptian japonica, indica, and indica/japonica genotypes, and to identify, DNA fingerprinting with employing simple sequence repeat (SSR) markers to develop DNA barcode. This study includes 15 rice genotypes that were genetically evaluated using 32 microsatellites. DNA barcodes as well as the unique pattern of SSR polymorphism were produced. A total of 116 alleles was detected and the number of alleles ranged from 2 to 5, with an average of 3.625 alleles per locus. The polymorphism information content (PIC) introduced a mean of 0.566 and there was a positive correlation between the highest value of PIC and the highest number of detected alleles by SSRs. The principal coordinate analysis, specifically, PC1 and PC2, explained 54.72% and 12.96% of the total variability, respectively, for the 32 SSR. One set of multiplex assay with five markers each (RM307, RM317, RM470, RM412 and RM242) was developed for all the 15 rice genotypes. The findings revealed that increasing genetic diversity in the national rice breeding programme requires introducing germplasm from other places with various genetic backgrounds. Furthermore, the identification, DNA barcode approach and certification of genotypes utilizing microsatellite markers could be a decent supplement to existing agro-morphological features when genotypes are firmly related.