Abstract: Grass carp (Ctenopharyngodon idellus) has been one of the most important traditional domestic freshwater aquaculture fish in China. Its habitat includes major rivers and lakes in China, but it is most abundant in Yangtze River and its adjacent lakes. In the present study, the genetic diversity and population structure of grass carp from Yangtze River system were investigated using microsatellite DNA markers. In total 109 individuals were collected from four sites of the Yangtze River System including Yibin, Sichuan; Jiayu, Hubei; Dongting Lake, Hunan and Poyang Lake, Jiangxi. A full-sib family was prepared using artifical fertilization. Genomic DNA was extracted from ethanol-preserved fin tissues by using phenol-chloroform method. Using a set of microsatellite primers originally reported in common carp (Cyprinus carpio), we carried out many cross-species PCR amplifications. PCR products were separated on 7.5% nondenaturing polyacrylamide gels, stained with ethidium bromide in water and visualized with ultraviolet. A successfully amplified locus with clear banding pattern (i.e. one or two bands) was subject to test for genetic segregation in a full-sib grass carp family before it was considered to be used in population analysis. Purified PCR products were cloned into E. coli DH5α strain using pMD-18T vector, and positive clones were selected and sequenced by Shanghai BioAsia Biotech Company for confirmation of repeat motifs. The size of alleles was scored by comparison with pBR322 DNA/Msp I markers. Five microsatellite primers successfully amplified six loci with reasonable polymorphism in grass carp. The number of alleles in these loci ranged from 3 to 7. These six microsatellite loci were inherited with Mendelian mode in a test full-sib grass carp family. Sequencing analysis for the PCR products in grass carp confirmed that all these six loci contained core repeat motifs, and some di-nucleotide motifs were similar to those in common carp (e.g. CA repeat) while others were new in grass carp, especially those tri-nucleotide repeats (i.e. TTA and GGA). Six microsatellite loci in grass carp, i.e. MFW1-1, MFW1-2, MFW5, MFW15, Koi3 and Koi20, were applied to subsequent studies on genetic diversity and possible population structure of grass carp samples from the Yangtze River System including river and its major adjacent lakes. The results showed that the number of alleles of a population ranged from 3.8 to 4.8 on average, and mean observed heterozygosity (Ho) from 0.40 to 0.57, while mean expected heterozygosity (H E) from 0.48 to 0.65. After applying Hardy-Weinberg Equilibrium (HWE) test, several loci were found to be significantly deviated from HWE in some populations in which deficiency of heterozygotes was apparent. Genetic distance analysis showed that the largest distance was between Sichuan and Dongting Lake population and the smallest distance was between Jiayu and Poyang Lake population. However, the analysis of molecular variance (AMOVA) indicated that almost majority of the variance in the grass carp was within populations (95.60%), and only small proportion was among populations (4.40%). The lower level of overall genetic diversity and particularly lower Fixation Index (Fst=0.044) indicated that the grass carp in Yangtze River system had a relatively lower genetic variation and its genetic structure between populations was weak.