Abstract We sought to determine if TNF promoter variants could explain iron phenotype heterogeneity in adults with previous HFE genotyping. HEIRS Study participants genotyped for C282Y and H63D were designated as high transferrin saturation (TS) and/or serum ferritin (SF) (high TS/SF), low TS/SF, or controls. We grouped 191 C282Y homozygotes as high TS/SF, low TS/SF, or controls, and 594 other participants by race/ethnicity as high TS/SF or controls. Using denaturing high-performance liquid chromatography (DHPLC), we screened the TNF promoter region in each participant. We performed multiple regression analyses in C282Y homozygotes using age, sex, HEIRS Study Field Center, and positivity for TNF − 308G→A and − 238G→A to determine if these attributes predicted ln TS or ln SF. DHPLC analyses were successful in 99.3% of 791 participants and detected 9 different variants; TNF − 308G→A and − 238G→A were the most prevalent. Most subjects positive for variants were heterozygous. The phenotype frequencies of each variant did not differ significantly ( p < 0.05) across subgroups of C282Y homozygotes, or across white, black, Hispanic, and Asian non-C282Y homozygotes subgrouped as high TS/SF phenotypes and controls. TNF − 308G→A positivity was a significant predictor of initial screening ln TS but not ln SF; TNF − 238G→A predicted neither ln TS nor ln SF. We conclude that TNF promoter variants have little, if any, effect on initial screening SF values in adults with or without C282Y homozygosity. We cannot exclude a possible association of homozygosity for TNF promoter variants on TS and SF values.