Myeloid differentiation 2 (MD-2) plays a critical role in orchestrating the innate immune response and the development of sepsis and subsequent organ dysfunction after trauma. The objectives of this prospective study were to identify haplotype tag single-nucleotide polymorphisms (htSNPs) within the entire MD-2 gene and to investigate their clinical relevance in patients with major trauma. A total of 726 patients with major trauma were prospectively recruited and composed of two different geographic populations (Chongqing in southwestern China and Zhejiang in eastern China). The htSNPs of the MD-2 gene were determined using HapMap database and linkage disequilibrium analysis. The htSNPs were genotyped using pyrosequencing method. The whole peripheral blood samples obtained immediately after admission were stimulated with bacterial lipopolysaccharide and then determined for production of tumor necrosis factor α. Sepsis morbidity rate and multiple organ dysfunction (MOD) scores were accessed. Three SNPs (rs7843858, rs11465996, and rs2114169) were identified as htSNPs for the MD-2 gene. All of them were shown to be high-frequent SNPs in this study cohort. However, only the rs11465996 polymorphism was shown to be significantly associated with higher sepsis morbidity rate and MOD scores in patients with major trauma in both Chongqing and Zhejiang districts. In addition, the rs11465996 polymorphism was significantly associated with tumor necrosis factor α production by peripheral blood leukocytes in response to bacterial lipoprotein stimulation. Among the three htSNPs of the entire MD-2 gene, only the rs11465996 might be used as relevant risk estimate for the development of sepsis and MOD syndrome in patients with major trauma.