Internal transcribed spacers in ribosomal DNA were often used as molecular sequence in phylogenetic study. The complete sequence of internal transcribed spacers of Macrobrachium rosenbergii was isolated by PCR. The length of ITS1 was 1 070-1 150 bp with a GC content of 51.4%-52.7%, and the length of 5.8S was 163 bp with a GC content of 55.2%. Compared to ITS1, the length of ITS2 (484-545 bp) was shorter while GC content (53.4%-54.6%) was higher. By comparison of sequence from different individuals, 61 SNP loci were found in ITS1, including 28 C/T transitions (45.9%), 23 A/G transitions (37.7%), 1 A/T transversions (1.6%), 3 T/G transversions (4.9%), 4 C/G transversions (6.6%), and 2 A/C transversions (3.3%); 15 SNP loci were found in ITS2, including 6 C/T transitions (40%), 5 A/G transitions (33.3%), 1 A/T transversions (6.7%), 2 T/G transversions (13.3%), and 1 C/G transversions (6.7%). SSR loci were searched by the SSRHunter software, and the results showed that the SSR loci in ITS1 and ITS2 were 14 and 6 respectively, the type of (AG)n was rich in ITS1 and the type of (GA)n was rich in ITS2.There were three repeat units with a length of 59 bp in ITS1, and the loci were 181, 390 and 781, respectively. The results of ITS intravariances analysis demonstrated that clones from the same individual were different in length because of SSR. Furthermore, homology comparison of ITS sequence indicated that the similarity of each ITS1 was 45.39%-50.64%, and the highest occurred between M. rosenbergii and M.hainanense , while the lowest occurred between M.rosenbergii and Exopalaemon carinicauda . 5.8S showed very high similarity with other crustaceans, and the homology was 100% between M.rosenbergii and M.nipponense . The similarity of each ITS2 was 42.53%-53.46%, and the highest occurred between M.rosenbergii and M.nipponense , while the lowest occurred between M.rosenbergii and Procambarus clark . Phylogenetic tree based on the ITS was constructed by Neighbor-joining method, and the results showed that the relationship between M.rosenbergii and M.nipponense was the closest, which accorded with morphology classification.