With the construction of a library of partial fractionated genomic DNA of Panulirus stimpsoni Hoehuis, the microsatellite sequences of P. stimpsoni were screened by PCR technique. Then, the genetic diversity was analyzed with the microsatellite markers. Seventy-eight microsatellite sequences in 55 positive recombinant clones were obtained by PCR technique with primers of M13+/- and (CT)15, and (AT)15. Among these microsatellite sequences, the numbers of perfect, imperfect, compound perfect, and compound imperfect sequences were 50 (64%), 3 (3.8%), 5 (7.7%), and 19 (24.5%), respectively. To analyze genomic DNA diversity of P. stimpsoni, 15 pairs of primers were designed from the microsatellite flanking sequences. In these microsatellite loci, the alleles numbers ranged from 3 to 12; and the sizes of these alleles ranged from 78 to 425 bp, which are in accordance with their predicted size range. The expected heterozygosity (He) and the polymorphism information content (PIC) ranged from 0.48 to 0.87 and 0.44 to 0.84 with the average values of 0.71 and 0.60, respectively. These results showed that these microsatellite loci were suitable for P. stimpsoni molecule markers and genetic analysis because of their richness in genetic information.