Abstract: Pyrosequencing is a new DNA sequencing method, it was used to analyze single nucleotide polymorphisms recently years. Cathepsin-L gene is a lysosomal protease gene that is involved in the intermolt cycle of shrimp. In this study, genotyping of cathepsin-L gene (GenBank accession no. AY366355) SNP (C681G) in 96 Litopenaeus vannamei shrimps was performed using Pyrosequencing. The C/C,C/G and G/G genotype were found in this population, and the frequency were 0.81, 0.16 and 0.03, respectively. The frequency distribution was consistent with the law of Hardy-Weinberg. The statistic analyses showed that C681G SNP had no significantly association BW in this population (P>0.05)．The results of this study showed that pyrosequencing is a versatile technique that could improve the efficiency of SNP analysis for shrimp genomic research.