Abstract: Microsatelliteenhanced genomic library of the tiger shrimp Penaeus monodon was constructed using repeatenrichment method with biotin-labeled oligos ［(CA)12, (AG)12 and (TA)16］ and streptavidin magnetic beads. From a total of randomly selected 254 clones, 51 clones (20.1%) were positive after PCR selection. Of which, 32 clones comes from (CA)12 probe used library, the left from (AG)12 probe-used library 48 clones are discovered to contain microsatellites. By alignment, 40 special microsatellite clones are confirmed finally (GA)n and (CA)n were the abundant microsatellite motifs with a percentage of 20.7% and 60.4% in the isolated microsatellites. Several other types of repeat sequences were detected, including repeats consisting of (AT)n, (GC)n, (TGG)n, (AAG)n, (AAT)n, (GAA)n, (GTGC)n, (GCGT)n, (GGTTA)n, (GTGCGT)nmaking up to 18.9%. Characterization of these microsatellites showed that 76 (68.5%) sequences were classified as perfect type, 22 (19.8%) as imperfect type and 13 (11.7%) as compound type. The repeat units of (GT/CA)n ranged from 3 to 52 was much more than the units of (GA/CT)n ranged from 3 to 27. The sequence size ranges from 129 to 601 bp with an average of 286 bp. This study provides a base for molecular breeding and assessment of germplasm resources of the tiger shrimp in future.