Abstract: The giant freshwater prawn (Macrobrachium rosenbergii) is one of the most important crustaceans in inland aquaculture, especially in many tropical and subtropical areas. China has become the largest producer of the giant freshwater prawn in the world. For protecting species resources and then achieving selective breeding, it is necessary to study the effect of artificial culture and selective breeding on the genetic diversity of M. rosenbergii. In this study, the ribosomal internal transcribed spacer 2 (ITS2) of 111 individuals from 5 populations (Bengal wild population, Burma wild population, Zhejiang culture population, Guangxi culture population and selected population “South Tailake No. 2”) were sequenced and analyzed in order to estimate the genetic diversity and genetic differentiation of M. rosenbergii. Genomic DNA was isolated following the standard method from muscle tissue, and then was kept at -20℃. PCR primers were synthesized by Shanghai Generay Biotech Co., Ltd. The PCR reaction mixture contained 25 μL, thermal cycling consisted of an initial denaturing at 94℃ for 2min, followed by 30 cycles of denaturing at 94℃ with each cycle of 30s, 45s annealing at 55℃, and 1min extension at 72℃, and concluded with a final extension at 72℃ for 10min. The PCR products, which were purified and sequenced by Shanghai Generay Biotech Co., Ltd and analyzed using CLUSTL W, Dna SP 4.10, Mega 4.0 and Arlequin 3.0 softwares.The results showed that 58 variations and 56 haplotypes were revealed in the 111 sequences. The mean contents of A, T, C and G were 21.6%, 24.4%, 19.5% and 34.6% in ITS2, and the content of G + C was significantly higher than A + T. It was also found that Bengal wild population had the highest genetic diversity with 7.186 for the average of nucleotide differences (K) and 0.0155 for nucleotide diversity (Pi). In decreasing order of genetic diversity, the others were Burma wild population, Zhejiang culture population, Guangxi culture population in turn. And selected population “South Tailake No. 2” had the lowest genetic diversity (K and Pi were 3.032 and 0.0065, respectively) among the five populations of M. rosenbergii. Pairwise Fst of control region sequence analysis among five populations showed that there were significant differences between the wild populations and the culture populations (P0.01). Significant differences were also found not only between selected population “South Tailake No. 2” and wild populations, but also between selected populations “South Tailake No. 2” and Guangxi culture population (P0.01). Genealogical tree showed that Bengal wild population and Burma wild population clustered together, Zhejiang culture population, Guangxi culture population and selected population “South Tailake No. 2” clustered together. The results indicated that the ITS2 sequence was a good molecular marker for detecting genetic variation of M. rosenbergii populations, in addition, artificial culture and breeding had led to decreases in genetic diversity and significant genetic differentiation among M. rosenbergii populations. After a 4-generation selection, there was clear genetic differentiation between new selected population “South Tailake No. 2” and the base population, and the new breed with stable characteristics would lay a foundation for breeding other new varieties.