Iodine is an essential micronutrient for thyroid hormone production. Adequate iodine intake and normal thyroid function are important during early development, and breastfed infants rely on maternal iodine excreted in breast milk for their iodine nutrition. The proportion of women in the United States of childbearing age with urinary iodine concentration (UIC) <50 μg/L has been increasing, and a subset of lactating women may have inadequate iodine intake. UIC may also be influenced by environmental exposure to perchlorate and thiocyanate, competitive inhibitors of iodine transport into thyroid, and lactating mammary glands. Data regarding UIC in U.S. lactating women are limited. To adequately assess the iodine sufficiency of lactating women and potential associations with environmental perchlorate and thiocyanate exposure, we conducted a multicenter, cross-sectional study of urinary iodine, perchlorate, and thiocyanate concentrations in healthy U.S. lactating women.Lactating women ≥18 years of age were recruited from three U.S. geographic regions: California, Massachusetts, and Ohio/Illinois from November 2008 to June 2016. Demographic information and multivitamin supplements use were obtained. Iodine, perchlorate, and thiocyanate levels were measured from spot urine samples. Correlations between urinary iodine, perchlorate, and thiocyanate levels were determined using Spearman's rank correlation. Multivariable regression models were used to assess predictors of urinary iodine, perchlorate, and thiocyanate levels, and UIC <100 μg/L.A total of 376 subjects (≥125 from each geographic region) were included in the final analyses [mean (SD) age 31.1 (5.6) years, 37% white, 31% black, and 11% Hispanic]. Seventy-seven percent used multivitamin supplements, 5% reported active cigarette smoking, and 45% were exclusively breastfeeding. Median urinary iodine, perchlorate, and thiocyanate concentrations were 143 μg/L, 3.1 μg/L, and 514 μg/L, respectively. One-third of women had UIC <100 μg/L. Spot urinary iodine, perchlorate, and thiocyanate levels all significantly positively correlated to each other. No significant predictors of UIC, UIC <100 μg/L, or urinary perchlorate levels were identified. Smoking, race/ethnicity, and marital status were significant predictors of urinary thiocyanate levels.Lactating women in three U.S. geographic regions are iodine sufficient with an overall median UIC of 143 μg/L. Given ubiquitous exposure to perchlorate and thiocyanate, adequate iodine nutrition should be emphasized, along with consideration to decrease these exposures in lactating women to protect developing infants.