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)_n making 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.