Context: Thyroglobulin (Tg) could be a sensitive biomarker of iodine nutrition in pregnant women (PW). A dried blood spot (DBS) assay would simplify collection and transport in field studies. Objectives: Our aims were to (1) establish and test a reference range for DBS-Tg in PW; (2) determine whether co-measurement of Tg antibodies (Abs) is necessary to define population iodine status. Design, Setting, and Participants: Standardized cross-sectional studies of 3870 PW from 11 countries. For the DBS-Tg reference range, we included TgAb-negative PW (n = 599) from 3 countries with sufficient iodine intake. Main Outcome Measures: We measured the urinary iodine concentration and DBS thyroidstimulating hormone, total thyroxin, Tg, and TgAb. Results: In the reference population, the median DBS-Tg was 9.2 g/L (95% confidence interval, 8.7 to 9.8 g/L) and was not significantly different among trimesters. The reference range was 0.3 to 43.5 g/L. Over a range of iodine intake, the Tg concentrations were U-shaped. Within countries, the median DBS-Tg and the presence of elevated DBS-Tg did not differ significantly between all PW and PW who were TgAb-negative. Conclusions: A median DBS-Tg of 10 g/L with <3% of values 44 g/L indicated population iodine sufficiency. Concurrent measurement of TgAb did not appear necessary to assess the population iodine status.