Using pooled DNA genotyping to estimate the proportional contributions from multiple families in a pooled sample is of particular interest for selective breeding in aquaculture. We compared different pooled libraries with separate 2b-RAD sequencing of Litopenaeus vannamei individuals to assess the effect of different population structures (different numbers of individuals and families) on pooled DNA sequencing, the accuracy of parent sequencing of the DNA pools and the effect of SNP numbers on pooled DNA sequencing. We demonstrated that small pooled DNA genotyping of up to 53 individuals by 2b-RAD sequencing could provide a highly accurate assessment of population allele frequencies. The accuracy increased as the number of individuals and families increased. The allele frequencies of the parents from each pool were highly correlated with those of the pools or the corresponding individuals in the pool. We chose 500-28,000 SNPs to test the effect of SNP number on the accuracy of pooled sequencing, and no linear relationship was found between them. When the SNP number was fixed, increasing the number of individuals in the mixed pool resulted in higher accuracy of each pooled genotyping. Our data confirmed that pooled DNA genotyping by 2b-RAD sequencing could achieve higher accuracy than that of individual-based genotyping. The results will provide important information for shrimp breeding programs.