There is no scientific consensus on whether breast milk iodine concentration (BMIC) accurately reflects iodine status in lactating mothers and breastfed infants. This study aimed to compare BMIC and maternal urinary iodine concentration (UIC) as indicators of iodine status in breastfed neonates. In this cross-sectional study, 147 lactating mothers and their neonates (3-5 days postpartum) were randomly selected from health care centers. Breast milk and urine samples were collected from each mother and neonate, and a heel-prick blood sample was taken from all neonates as part of a congenital hypothyroidism screening program. According to the World Health Organization criteria, median urinary iodine concentration (UIC) ≥ 100 μg/L in lactating mothers and neonates indicates iodine sufficiency. In areas of iodine sufficiency, median BMIC ≥ 100 μg/L is considered an adequate level. Overall, 129 (89.0%) and 16 (11.0%) mothers had BMICs ≥ 100 and ˂ 100 μg/L, respectively. Median (interquartile range [IQR]) maternal UIC was 70 μg/L (42-144 μg/L) and 37 μg/L (25-100 μg/L) in mothers with breast milk iodine levels ≥ 100 and ˂ 100 μg/L, respectively (P = 0.047); values for UIC of neonates born to mothers with BMICs ≥ 100 and ˂ 100 μg/L were 230 μg/L (114-310 μg/L) and 76 μg/L (41-140 μg/L), respectively (P < 0.001). In the linear regression model, neonate UIC was positively associated with BMIC in both unadjusted (β = 0.558, P < 0.001) and adjusted analysis (β = 0.541, P < 0.001). A similar result was found in logistic regression analysis, indicating that neonates born to mothers with BMIC ≥ 100 μg/L were more likely to have UIC ≥ 100 μg/L compared to those whose mothers had BMIC < 100 μg/L in both unadjusted (OR = 7.93, P < 0.001) and adjusted analysis (OR = 7.29, P = 0.001). The present findings indicate that BMIC is a more sensitive indicator than maternal UIC for assessment of iodine status in breastfed neonates. To address low levels of maternal UIC, further studies on the prescription of supplements containing 150 μg/day iodine during lactation period are warranted.