Iodine nutrition is commonly assessed from iodine excretion in urine. A 24 h urine sample is ideal, but it is cumbersome and inconvenient. Hence, spot urine samples with creatinine to adjust for differences in void volume are widely used. Still, the importance of ethnicity and the timing of spot urine samples need to be settled. We, thus, collected 104 early morning spot urine samples and 24 h urine samples from Inuit and non-Inuit living in Greenland. Diet was assessed by a FFQ. Demographic data were collected from the national registry and by questionnaires. Iodine was measured using the Sandell–Kolthoff reaction, creatinine using the Jaffe method and para-amino benzoic acid by the HPLC method for the estimation of completeness of urine sampling and compensation of incomplete urine samples to 24 h excretion. A population-based recruitment was done from the capital city, a major town and a settlement (n 36/48/20). Participants were seventy-eight Inuit and twenty-six non-Inuit. The median 24 h iodine excretion was 138 (25th–75th percentile 89–225) μg/97 (25th–75th percentile 72–124) μg in Inuit/non-Inuit (P= 0·030), and 153 (25th–75th percentile 97–251) μg/102 (25th–75th percentile 73–138) μg (P= 0·026) when including compensated iodine excretion. Iodine excretion in 24 h urine samples increased with a rising intake of traditional Inuit foods (P= 0·005). Iodine excretion was lower in morning spot urine samples than in 24 h urine samples (P< 0·001). This difference was associated with iodine intake levels (P< 0·001), and was statistically significant when the iodine excretion level was above 150 μg/24 h. In conclusion, the iodine intake level was underestimated from morning spot urine samples if iodine excretion was above the recommended level.