Due to its high tolerance to salinity and temperature, as well as its strong adaptability, Metapenaeus ensis holds an important position in the Chinese aquaculture industry. However, studies on the evaluation of its germplasm resources remain insufficient. This research conducted an in-depth comparative evaluation of M. ensis from three representative farming regions in China: Sanya, Zhuhai, and Raoping. The nutritional analysis of muscle tissue showed no statistically significant differences in crude ash, moisture, and crude protein content among the populations (p > 0.05). However, significant differences were observed in crude fat and total sugar content (p < 0.05). The MeSY and MeRP populations had higher crude fat content than the MeZH population (p < 0.05), while the MeZH population exhibited the highest total sugar content. In terms of amino acid composition, the MeSY population had relatively higher total essential amino acid content and proportion, as well as higher total amino acid content, both of which were statistically significant (p < 0.05). A fatty acid composition analysis further highlighted the advantages of the MeRP population in several key fatty acids (p < 0.05). Physiological and biochemical analyses showed no significant differences among the three populations in total antioxidant capacity, superoxide dismutase activity, or catalase activity (p > 0.05). A genetic diversity analysis indicated that M. ensis has relatively low diversity, with the MeSY population showing higher SNP density and nucleotide diversity. A genetic differentiation analysis revealed significant genetic differentiation between the MeSY and MeZH populations, while differentiation between the MeZH and MeRP populations was relatively smaller. This comprehensive assessment of nutritional components, amino acids, fatty acids, antioxidant capacity, and genetic diversity highlights the advantages of germplasm resources from different regions. These findings provide valuable insights for future research on the genetic characteristics and breeding potential of M. ensis.