Background: Lactating women (LW) and infants have high dietary iodine requirements and they are at risk for iodine deficiency.----- Aims: The aim is to assess iodine status and thyroid function in LW and their breastfed infants in Zagreb, Croatia. Methods: The study included 116 LW and 166 breastfed infant pairs from the City of Zagreb. Urinary iodine concentration (UIC) and thyroid function parameters (TSH, T4, Tg) from the blood were measured in all subjects, while iodine concentration in breast milk (BMIC) was additionally determined in LW. Additionally, data on the intake of foods containing iodine and salt were collected. ----- Results: Survey analysis indicated that 99,2% of the LW used iodized salt in household and 12,9% used iodine containing vitamin and mineral supplements at the time of the research. The median (IQR) of UIC was 84, 5 μg/L (26,0-149,7 μg/L) in LW and 231,2 μg/L (153,0-351,5 μg/L) in infants, whereas BMIC was 122 μg/L (88,0- 184,0 μg/L). A statistically significant positive correlation between BMIC and infants UIC (p < 0.001) as well as between LW and the infants’ thyroid function parameters were observed. ----- Conclusion: This is the first study in Croatia demonstrating that BMIC is a reliable biomarker of iodine status during lactation and could predicts iodine intake in infants. The study confirms that mandatory salt iodization with 25 mg KI/ kg of salt in Croatia ensures sufficient dietary iodine for LW and optimal iodine nutrition for breastfed infants through breast milk.