SSRs or microsatellites identification and its functional analysis has a key role in different sectors of genomics such as genome organization, gene regulation, quantitative genetic variation, evolution of genes and plant breeding sectors. Therefore, computational approach was undergone for identification and analysis of SSR within functional domain of Citrus limon (C. limon) of family Rutaceae, is one of the vital and effective medicinal plant. Total 1644 numbers of extracted ESTs of C. limon were validated through Tandem repeat finder and VecScreen which have been assembled in CAP3 that resulted 55 contigs and 1183 singletons. Afterwards, total 420 SSRs were identified as SSR-ESTs using MISA tool and also detected 75.23% of mononucleotide SSR motifs with most ample sort of repeats such as di- (9.52%), tri- (7.61 %) and tetra- (0.95 %) nucleotide. Ultimately, 128 SSR sequences have been selected with appropriate primer properties which would be used as markers to look at transferability to related species. Further, the useful functional annotation was performed using Blast2GO. These findings would assist to understand the significance of SSR markers and also to facilitate the evaluation of genetic range in medicinal plant flora.