The liver, as the major organ for iron storage and production of hepcidin, plays pivotal roles in maintaining mammalian iron homeostasis. A previous study showed that Quantitative Trait Loci (QTLs) on chromosome 7 (Chr7) and 16 (Chr16) may control hepatic non-heme iron overload in an F2 intercross derived from C57BL/6J (B6) and SWR/J (SWR) mice. In this study, we aimed to validate the existence of these loci and identify the genes responsible for the phenotypic variations by generating congenic mice carrying SWR chromosome segments expanding these QTLs (D7Mit68-D7Mit71 and D16Mit125-D16Mit185, respectively). We excluded involvement of Chr7 based on the lack of iron accumulation in congenic mice. In contrast, liver iron accumulation was observed in Chr16 congenic mice. Through use of a series of subcongenic murine lines the interval on Chr16 was further fine-mapped to a 0.8 Mb segment spanning 11 genes. We found that the mRNA expression pattern in the liver remained unchanged for all 11 genes tested. Most importantly, we detected 4 missense mutations in 3 candidate genes including Sidt1 (P172R), Spice1(R708S), Boc (Q1051R) and Boc (S450-insertion in B6 allele) in the liver of SWR homozygous congenic mice. To further delineate potential modifier gene(s), we reconstituted seven candidate genes, Sidt1, Boc, Zdhhc23, Gramd1c, Atp6v1a, Naa50 and Gtpbp8, in mouse liver through hydrodynamic transfection. However, we were unable to detect significant changes in liver iron levels upon reconstitution of these candidate genes. Taken together, our work provides strong genetic evidence of the existence of iron modifiers on Chr16. Moreover, we were able to delineate the phenotypically responsible region to a 0.8 Mb region containing 11 coding genes, 3 of which harbor missense mutations, using a series of congenic mice.