Cold tolerance is one of the major economic characters in fish. In order to discuss the cold tolerance of large yellow croaker (Pseudosciaena crocea R.), fifteen fluorescent dye-labeled microsatellite markers were applied to detect genetic differences between F1 offsprings of cold tolerance group and normal group of large yellow croaker by SSR-PCR. Each group contained 20 randomly and separately sampled individuals. As a result, marker LYC0002 five alleles (LYC0002(104 bp), LYC0002(106 bp), LYC0002(108 bp), LYC0002(110 bp), and LYC0002(112 bp)) were amplified with marker LYC0002 in both groups and 60% (12/20) of individuals had allele LYC0002(112 bp) in cold tolerance group exclusively, which indicated that this allele is probably sensitive to temperature and associated with gene for cold tolerance. In addition, four alleles (LYC0002(106 bp), LYC0002(108 bp), LYC0002(110 bp), and LYC0002(112 bp)) were sequenced individually. Sequence alignments showed that LYC0002(112 bp) allele contains 10 (CA) repeats, the remaining three alleles lacked one (CA) one by one, corresponding to the stepwise mutation model (SMM) of microsatellite.