In order to evaluate the genetic parameters of body length, weight and WSSV resistance of Litopenaeus vannamei more accurately, 8 microsatellite loci were used to genotype mother and offspring of 69 families of L. vannamei, and the pedigree reconstruction and the calculation of the molecular relationship correlation were carried out by using the genotyping information. The reconstructed pedigree, the molecular affinity and the physical pedigree were used to construct the additive genetic correlation matrix. Then, the genetic parameters of body length, body weight and WSSV resistance were evaluated. Finally, through the cross validation method, the prediction ability and accuracy of genetic parameter evaluation were compared. The results of genotyping showed that 166 alleles were detected in 8 microsatellite loci; the number of alleles in each locus was 12-45; the average number of alleles in each locus in parent and offspring population was 9.50 and 18.13; the average observed heterozygosity was 0.618 and 0.709; the average expected heterozygosity was 0.749 and 0.775; the average polymorphism information content was 0.711 and 0.750, respectively, and all of loci showed high polymorphism. The heritability of reconstructed pedigree, molecular affinity and physical pedigree analysis showed that the heritability of body length was 0.119±0.031, 0.120±0.032, 0.132±0.030; the heritability of body weight was 0.176±0.039, 0.182±0.040, 0.172±0.034; and the heritability of resistance to WSSV was 0.135±0.033, 0.134±0.033 and 0.098±0.031, respectively. In the heritability of body length, body weight and resistance to WSSV, the standard deviation of molecular affinity was smaller than that of reconstructed pedigree and physical pedigree. Through cross validation, the prediction ability and accuracy of the three genetic parameters evaluation from high to low are molecular affinity, reconstructed pedigree and physical pedigree. The results showed that it was more accurate to evaluate the body length, body weight and WSSV resistance of L. vannamei by using microsatellite molecular markers than using physical pedigree. In the method of evaluating heritability by using microsatellite molecular markers, it was more effective to directly use the genetic correlation of offspring. This study provides a more accurate method for the evaluation of genetic parameters of L. vannamei and a reference for further breeding.