Prenatal exposure to total mercury (T-Hg) comes from both natural and anthropogenic sources. T-Hg can cross the blood-brain and placental barriers, and may be associated with future neurological and physiological dysfunctions. Scalp hair is an optimal and non-invasive indicator of chronic T-Hg exposure. As part of the Maternal-Infant Research on Environmental Chemicals (MIREC) Study, hair samples from 350 women were collected within weeks after giving birth, to determine temporal variations in T-Hg levels from preconception to delivery, and to compare these levels to corresponding levels measured in other matrices (maternal and umbilical cord blood, and infant’s meconium). A maximum of 12 one-cm hair segments were cut starting at the scalp; segments closer to the scalp reflected recent exposure (within the last month). For proper comparison, the hair segments were matched with the collection dates for other matrices. GM hair T-Hg levels greatly decreased during pregnancy, from 0.26 μg g−1 (preconception or full-length hair) to 0.18 μg g−1 (at delivery or segments closer to the scalp). A similar decreasing trend was found for T-Hg in maternal blood: 1st trimester (0.60 μg L−1) to 3rd trimester (0.47 μg L−1). The median hair-to-blood ratios of T-Hg levels varied from 364 (1st trimester), to 408 (3rd trimester), to 229 (cord blood). Very low T-Hg levels were detected in meconium. Mercury levels in blood and hair correlated with consumption of large predatory fish.