Catharanthus roseus (periwinkles) belongs to the Apocynaceae family with great anti-cancer, anti-diabetic, and hepatoprotective values. Due to the large number of active molecules accumulated in these plants, they are of particular concern, especially in the pharmaceutical sector. The availability of ESTs gave the genetic algorithm of the plant to differentiate between the species accessions at the genetic level. The high-throughput method used for mining and detection of microsatellites (SSRs) embedded in ESTs gave a new insight for molecular markers’ development. 19899 ESTs were retrieved, examined by NCBI EST dB and assembled 2692 to get full-length contigs sequences. 338 microsatellites (SSR) loci were predicted with an average of SSR per 9.33 kb of EST though MISA-web tools out of 2692 contigs. Furthermore, trinucleotide, a well-known SSR was examined and found to be the most favorable repeats' type (26.62%) followed by dinucleotide (24.22), mononucleotide (48.22%), and hexanucleotide (0.3%) types. The highest frequency of (A/T)n was reported in this finding followed by (AAG)n. The simple sequence repeats (SSR) extracted from C. roseus EST's data were used as molecular tools for genetic characterization in the present study. These predicted SSRs can be significantly used for constructing the genetic maps and also for differentiating the accession between the species.