Adequate breast milk iodine concentration (BMIC) is essential for the growth and cognitive development of exclusively breastfed infants; however, data on variations in BMIC over 24 h are limited. We aimed to explore in lactating women the variation in 24-h BMIC. Thirty pairs of mothers and breastfed infants aged 0-6 mo were recruited from the cities of Tianjin and Luoyang, China. A 3-d 24-h dietary record, including salt intake, was performed to assess the dietary iodine intake of lactating women. Breast milk samples before and after each feeding for 24 h and 24-h urine samples were collected from the women for 3 d to estimate iodine excretion. A multivariate linear regression model was used to analyze the factors influencing BMIC. A total of 2658 breast milk samples and 90 24-h urine samples were collected. The median BMIC and 24-h urine iodine concentration (UIC) of lactating women for a mean of 3.6 ± 1.48 mo were 158 μg/L and 137 μg/L, respectively. The interindividual variability of BMIC (35.1%) was higher than that observed within individuals (11.8%). The variation in BMIC showed a "V" shaped curve over 24 h. The median BMIC at 08:00-12:00 (137 μg/L) was significantly lower than that at 20:00-24:00 (163 μg/L) and 00:00-04:00 (164 μg/L). A progressively increasing curve was obtained for BMIC until it peaked at 20:00 and plateaued at a higher concentration from 20:00 to 04:00 than at 08:00-12:00 (all P < 0.05). BMIC was associated with dietary iodine intake (β: 0.366; 95% CI: 0.004, 0.018) and infant age (β: -0.432; 95% CI: -1.07, -0.322). Our study shows that the BMIC presents a "V" shaped curve over 24 h. We recommend that breast milk samples be collected between 08:00 and 12:00 for evaluation of the iodine status of lactating women.