The giant river-catfish Sperata seenghala is one of the commercially important freshwater catfish of India with wide distribution in all major rivers and reservoirs. The market demand is met through only from capture fisheries, so this may led to over exploitation of resources and subsequent stock depletion may transpire. Microsatellites are the most suitable markers for characterizing the genetic stocks. The present study was carried out to isolate microsatellite markers from S. seenghala collected from five major riverine systems of India, specifically Brahmaputra, Ganga, Godavari, Mahanadi and Narmada. Five repeat enriched genomic DNA libraries were prepared representing five populations. Each library includes DNA pooled from ten individuals. Both enzymatic (Ion Shear™ Plus Enzyme Mix II) and mechanical shearing (Covaris™ M220 Focused-ultrasonicator) was carried out to fragment the genomic DNA in to 350-400 bp size. Nineteen probes consisting di (3 no.), tri (7 no.), tetra (7 no.) and penta (2 no.) were designed to target di, tri, tetra and penta-nucleotide repetitive sequences. The size of probes was maintained between 24 to 27 bp and all the probes were labelled with biotin at 5' end. The Ion Torrent Personal Genome Machine® (PGM™) was used to sequence the SSR enriched DNA libraries of S. seenghala. The software MISA (MIcroSAtellite identification tool) (http://pgrc.ipk-gatersleben.de/misa/) was used to identify the repetitive sequences from the S. seenghala genome. A total of 17794 SSR loci were identified from all the populations. In all the populations the proportion of dinucleotide repeat motif was higher than any other type of motif i.e (45.44%). The percentage of tri and tetra nucleotide SSR were (8%) and (6 %) respectively. Although the contribution of penta and hexa nuecleotide repeat motifs were very trivial i.e (0.61% and 0.06%) respectively, but it was a very attention-grabbing finding as these type of repeat motifs are very important to develop stock specific markers. Lastly the contribution of compound SSR was very high i.e 21.83%.