This study aimed to evaluate the quality and genetic diversity of farmed Litopenaeus vannamei across three distinct populations from Maoming City (MM), Zhanjiang City (ZJ), and Yangjiang City (YJ) in Guangdong Province. Muscle tissues from L. vannamei were analyzed for phenotypic traits, conventional nutrients, amino acids, and fatty acids, while genetic diversity was assessed using whole genome sequencing techniques. The analysis revealed that the crude protein content in shrimp across the three populations ranged from 20.87 to 21.95 g/100 g, crude fat content ranged from 0.90 to 1.50 g/100 g, essential amino acid content ranged from 5.55 to 5.86 g/100 g, total amino acid content ranged from 14.73 to 15.27 g/100 g, total fatty acid content ranged from 682.73 to 793.97 mg/100 g, total antioxidant capacity (T-AOC) ranged from 2.68 to 2.72 μmol/g, superoxide dismutase (SOD) activity ranged from 1021.97 to 1057.21 U/g, and catalase (CAT) activity ranged from 78.65 to 81.33 μmoL/min. No significant differences were observed in ash and crude fat levels among conventional nutrients, nor in the biochemical indexes T-AOC, CAT, and SOD. Genetic analysis showed that the single nucleotide polymorphism density (SNP/Kb) ranged from 15.323 to 17.461, nucleotide diversity (π) ranged from 2.98 × 10-5 to 15.84 × 10-5, polymorphism information content (PIC) ranged from 0.300 to 0.317, heterozygosity (Ho) ranged from 0.033 to 0.048, and inbreeding coefficients (FIS) ranged from 0.834 to 0.887. The genetic differentiation index (FST) values among the three populations ranged from 0.056 to 0.106. This study provides an evaluation of the germplasm resources and genetic diversity of farmed L. vannamei, offering insights for the efficient management and sustainable utilization of this species' germplasm resources.