Grass carp (Ctenopharyngodon idella) 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. idella has important theoretical value and practical significance. Microsatellite markers are popular in breeding studies with characterization of wide distribution, large numbers and high polymorphism. In this study, microsatellite loci were mined based on whole genome sequences of C. idella 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 were found in the 900.51 Mb genome sequence of C. idella, with a total length of 12 835 407 bp, accounting for 1.425 4% of the whole genome length. The average distance of SSR was 1 329.43 bp. The mononucleotide repeats motifs (52.85%) were the most common, followed by 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 were A/T, AC/GT, AAT/ATT, AGAT/ATCT, AATAT/ATATT and AACCCT/AGGGTT, respectively. The distributions of copy numbers in different microsatellites repeat types showed there was a significant negative correlation between repeats motifs length and mean copy number (r = −0.8581, P = 0.0288). The copy numbers of mononucleotide repeats were focused mainly on 10-29 (98.11%), dinucleotide repeats 6-25 (96.80%), trinucleotide repeats 5-11 (95.14%), tetranucleotide repeats 5-10 (88.48%), pentanucleotide repeats 5-10 (84.93%), hexanucleotide repeats 5-7 (87.42%). 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. idella used as a 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. idella, screened out highly polymorphic microsatellite markers with 4-6 base repeats and carried our parental identification of C. idella 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. The study summarized the Characteristics of whole genome microsatellites of C. idella and laid a foundation for the application of C. idella microsatellites in its breeding program.