Objectives: To quantitatively assess the intra- and inter-individual variation of urinary iodine concentration in Japanese to determine whether urinary analysis is applicable to assessing habitual iodine intake in subjects on an individual basis. Methods: Five urine samples (first void) were taken from each of the 14 healthy female subjects at 2–3 week intervals over 4–5 months. Information on diet and medication use on the previous day of urine sampling was obtained by a questionnaire during each urine sampling. The concentration of iodine in urine samples was measured by inductively coupled plasma mass spectrometry (ICP-MS). Intra- and inter-individual variation was assessed by intra class correlation coefficient (ICC). Results: The median concentration of iodine in 70 urine samples was 91 μg/g-cre (range: 15–4400 μg/g-cre). The mean iodine concentration in urine samples from subjects who took iodine-rich foods/medications on the day before sampling was statistically significantly higher than that from subjects who did not take such foods/medications (p < 0.01, t-test). The ICC of urinary iodine concentration of the 14 subjects was 0.55, indicating good reproducibility; however, this was 0.28 when one subject who routinely used an iodine-containing gargle was excluded from analysis. Conclusions: Urine sampled on a single occasion is not a suitable medium for the assessment of long-term intake levels of iodine in subjects on an individual basis.