Aim: To evaluate the effect of collecting multiple (four) urine samples on the extensive variance often observed within a cohort when determining iodine status via urinary iodine concentration (UIC). Methods: Fifty-one children aged two to three years and thirty children aged 8–10 years participated in the study in South East Queensland, Australia. Each child's four urine samples were analysed using ammonium persulphate digestion before a Sandell–Kolthoff reaction method. Analysis of variance techniques were used to assess the effect of using multiple urine samples. Results: The median UICs were 223.3 and 141 μg/L for two- to three-year-olds and eight- to 10-year-olds, respectively. The coefficient of variance (CV) of UIC for children aged two to three years was reduced by 35.6%, 36.5% and 39.7% when two, three and four samples were included in the adjustment, respectively. Similarly, the CV of UIC for children aged 8–10 years was reduced by 24.7%, 30.7% and 34.7%, respectively. Conclusion: Although the practicality and cost of collecting multiple UICs need to be considered, collecting multiple UIC samples from each participant provides a more accurate reflection of a cohort iodine status.