Abstract: We constructed an (AC)n-microsatellite-enriched library for large yellow croaker Larimichthys crocea(Richardson 1846) using the method of FIASCO. Ninety clones were randomly selected for further colony amplification. The rate of positive clones reached to 66.7%, and 56 of the 60 positive clones sequences contained one or more SSR. By the analysis of repeat motif, dinucleotide repeats were the most dominant (91.1%), followed by trinucleotide repeats (8.9%). Among dinucleotides, (AC)n repeats were the most frequent (94.1%). Using Webers classification rules, the sequences were divided into three categories, the percentage of perfect repeat sequences, imperfect repeat sequences, and compound repeat sequences was 75.0%, 8.9%, and 16.1, respectively. Fifty two microsatellites primer pairs were designed and synthesized. The result showed that 35 SSRs were polymorphic in 46 F1 generation individuals. Twenty eight (80.0%) loci exhibited a segregation ratio of 1:1:1:1 (AB CD/AB AC), serving as the most useful markers segregating in co-dominant fashion. Six loci exhibited a segregation ratio of 1:1, and one loci exhibited a segregation of 1:2:1. Polymorphism analysis showed that 32 SSRs were consisted with Mendelian segregation ratio and could be used to construct the linkage map, while the other 3 SSRs (LYC0137, LYC0139, LYC0152) were departure from the expected Mendelian segregation patterns. These SSRs should be available for genetic linkage mapping, population genetics studies, molecular evolution and phylogenetic study of large yellow croaker.