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Pregnant women, lactating mothers, and their newborns constitute the target population for prevention and control of iodine deficiency. Hence, the aim of this study was to evaluate the iodine nutrition status among these vulnerable groups in an area with more than two decades of successful elimination of iodine deficiency. In this cross-sectional study conducted in health care centers of Tehran, 100 pregnant women and 84 lactating mothers and their newborn were randomly selected. Urinary iodine concentration and iodine content of salts were measured using the Sandell-Kolthoff and titration methods, respectively. Urinary iodine concentration <150 μg/L for pregnant women and <100 μg/L for lactating mothers and newborns was considered as iodine nutrition inadequacy, respectively. Median (interquartile range [IQR]) urinary iodine concentration (UIC) was 103 (59-155) μg/L in pregnant women, 77 (42-194) μg/L in lactating mothers, and 198 (84-260) μg/L in newborns. Median (IQR) iodine content of salt was 26 (21-30) ppm and 25 (18-28) ppm in pregnant women and lactating mothers, respectively (P = 0.462). Iodine content of salt was significantly correlated with UIC of pregnant women (r = 0.24, P = 0.019), but no correlation was found among lactating mothers (r = 0.12, P = 0.316). Neonatal UIC was significantly correlated with iodine content of salt consumed by their mothers (r = 0.49, P = 0.001). Despite suboptimal iodine status among subgroups of Tehranian pregnant and lactating women, iodine nutrition status of newborns was within optimal levels, which may be explained by a compensatory mechanism in the mammary glands.