Mandatory I fortification in bread was introduced in Australia in 2009 in response to the re-emergence of biochemical I deficiency based on median urinary I concentration (UIC)<100 µg/l. Data on the I status of lactating mothers and their infants in Australia are scarce. The primary aim of this study was to assess the I status, determined by UIC and breast milk I concentration (BMIC), of breast-feeding mothers in South Australia and UIC of their infants. The secondary aim was to assess the relationship between the I status of mothers and their infants. The median UIC of the mothers (n 686) was 125 (interquartile range (IQR) 76–200) µg/l and median BMIC (n 538) was 127 (IQR 84–184) µg/l. In all, 38 and 36 % of the mothers had a UIC and BMIC below 100 µg/l, respectively. The median UIC of infants (n 628) was 198 (IQR 121–296) µg/l, and 17 % had UIC<100 µg/l. Infant UIC was positively associated with maternal UIC (β 0·26; 95 % CI 0·14, 0·37, P<0·001) and BMIC (β 0·85; 95 % CI 0·66, 1·04, P<0·001) at 3 months postpartum after adjustment for gestational age, parity, maternal secondary and further education, BMI category and infant feeding mode. The adjusted OR for infant UIC<100 µg/l was 6·49 (95 % CI 3·80, 11·08, P<0·001) in mothers with BMIC<100 µg/l compared with those with BMIC≥100 µg/l. The I status of mothers and breast-fed infants in South Australia, following mandatory I fortification, is indicative of I sufficiency. BMIC<100 µg/l increased the risk of biochemical I deficiency in breast-fed infants.