Tumor necrosis factor alpha (TNFalpha) is a potent cytokine with a wide range of pro-inflammatory activities and plays a critical role in the pathogenesis of a number of infectious, inflammatory, autoimmune, and metabolic diseases. We determined four single-nucleotide polymorphisms (SNPs), -1031(C/T), -863(C/A), -857(C/T), and -308(G/A) in the TNFalpha promoter region using melting temperature analysis, among 1451 geriatric autopsy samples. Two adjacent SNPs, -863(C/A) and -857(C/T), were directly assayed by a single probe reaction, which correctly determined three of four expected haplotypes. Sequence confirmation related that the most rare haplotype (8/2902 chromosomes, frequency: 0.26%) contains a novel mutation of -856(G/A), instead of the predicted haplotype. These results indicate that melting temperature analysis provides a robust method to determine the polymorphisms in the TNFalpha promoter.