Iron homeostasis is tightly regulated and the peptide hormone hepcidin is considered to be a principal regulator of iron metabolism. Previous studies in a limited number of mouse strains found equivocal sex and strain dependent differences in mRNA and serum levels of hepcidin. Similarly, conflicting data on the relationship between hepcidin (Hamp1) mRNA levels and iron status as assessed by transferrin saturation and tissue iron concentrations were reported. Our aim was to clarify the relationships between strain, sex and hepcidin expression by examining multiple tissues and the effects of different dietary conditions in multiple inbred strains. Two studies were done: first, Hamp1 mRNA, liver iron and plasma diferric transferrin levels were measured in fourteen inbred strains on a control iron diet; and second, Hamp1 mRNA and plasma hepcidin levels in both sexes, as well as iron levels in the heart, kidneys, liver, pancreas and spleen in males, were measured in nine inbred/recombinant inbred strains raised on an iron sufficient or high iron diet. Both sex and strain have a significant effect on both hepcidin mRNA (primarily a sex effect) and plasma hepcidin levels (primarily strain effect). Hepatic hepcidin mRNA level is a highly significant predictor of plasma hepcidin levels. However, liver iron or diferric transferrin levels are not a predictor of Hamp1 mRNA levels in iron sufficient or high iron diet, nor are the Hamp1 mRNA and plasma hepcidin levels good predictors of tissue iron levels, at least in males.