The Hsp70 gene plays an important role in the animal immune response. In order to analyze the linkage between the genetic polymorphisms of Hsp70 and the virus-resistance trait of the shrimp, we detected SNPs in the coding region of Hsp70 by direct sequencing in 104 specimens of Litopenaeus vannamei from three shrimp populations (one TSV-resistance line and two TSV-susceptibility lines). Five SNPs were found including 661C/A, 712T/C, 782C/T, 892C/T, and 1090C/T in the fragment of Hsp70 (GenBank accession no. AY645906). There were significant difference of the allelic frequency of SNP 892C/T polymorphism between the TSV-resistance population and the two TSV-susceptibility populations (P < 0.001). A TSV-challenged test was performed to divide the shrimps into TSV-resistance group and TSV-susceptibility group. The 892C/T SNP genotypes of each shrimp sample were detected by pyro-sequencing. The allelic frequency of SNP 892C/T were significant difference between TSV-resistance group and TSV-susceptibility group (P < 0.001). The results show that the genetic polymorphisms of Hsp70 are likely associated with virus-resistance trait in populations of L. vannamei.