Enriched mini-genomic libraries of sugarcane variety VMC 87-599 were constructed using AatII and PstI restriction enzymes. A total of 517 sequences were obtained from the combined libraries. BLASTn homology revealed that majority (49%) of the sequences had no significant similarity whereas 22% had similarity with Saccharum officinarum, 12% with both Sorghum bicolor and Zea mays, 2% with Setaria italica, 1% with Oryza sativa, and 2% with other grass species. Also, 3.6% of the sequences had similar gene identity that is organellar and 12.7% nuclear. Of the total sequences, 54 (4.64%) had SSRs and 63 primers were developed with 11 primers mined from genic sequences. SSR repeat motifs were predominantly tetranucleotide (51%) and trinucleotide (30%). The primer sets were used to screen 20 sugarcane accessions of which 46 primers were amplified fragments. A total of 326 alleles were detected with the mean value of seven alleles per locus. The average PIC value of the 46 optimized primers ranged from 0 to 0.94 with a mean value of 0.73. Cluster analysis of genotypic data of 20 accessions from 46 primers revealed five clusters at a dissimilarity coefficient of 0.5. SSR markers designed from the enriched library. These are highly informative and can be utilized for sugarcane genetics and breeding.