| Grass carp (Ctenopharyngodon idellus) is the most productive freshwater aquaculture species in China which is of great importance for Chinese aquaculture industry. Developing molecular marker assisted breeding program for C. idellus has important theoretical value and practical significance. Microsatellite markers are popular in breeding studies with characterized of wide distribution, large numbers and high polymorphism. In this study, microsatellite loci were mined based on whole genome sequences of C. idellus and the distribution characteristics of the loci were analyzed. In the meantime, microsatellite markers with 4-6 bases repeats with high polymorphism and high accuracy of paternity test were developed. The results showed that 677, 363 microsatellite sequences are found in the 900.51 Mb genome sequence of C. idellus, with a total length of 12, 835, 407 bp, accounting for 1.425 4% of the whole genome length. The average distance of SSR is 1, 329.43 bp. The mononucleotide repeats motifs (52.85%) are the most common, following dinucleotide repeat (31.44%), tetranucleotide repeat (8.05%), trinucleotide repeat (6.47%), pentanucleotide repeat (1.12%), hexanucleotide repeat (0.07%). The preponderance of 1-6 base repeat motifs are A/T, AC/GT, AAT/ATT, AGAT/ATCT, AATAT/ATATT and AACCCT/AGGGTT, respectively. The 110 loci with 4-6 base repeats were randomly selected and primers were designed. Among these loci, 50 loci were amplified successfully with mixed DNA of C. idellus used as template and 15 loci showed high polymorphsim when analyzed in its breeding parent population. Cervus 3.0 analysis results showed that average expected heterozygosity of 15 loci was 0.802 ~ 0.959. The accuracy of simulated paternity test was 100% with 95% confidence degree. These 15 loci were used to identify 192 offspring from 20 families, and all offspring matched to the correct parents successfully with the accuracy of identification being 100%. In this study, we analyzed the microsatellite characteristics of C. idellus, screened out highly polymorphic microsatellite markers with 4-6 base repeats and achieving parental identification of C. idellus by using these highly polymorphic microsatellite loci. At the same time, it was proved that 4-6-base repeat microsatellite is more suitable for paternity test than traditional 2-3-base repeat. This study lays a foundation for the application of C. idellus microsatellites in its breeding program.