Objective:To investigate the iodine nutritional status of adults and pregnant women in Jianshui County and Zhenxiong County, Yunnan Province.Methods:From May to August 2021, one urban area and one rural area in Jianshui County and Zhenxiong County, Yunnan Province were selected as investigation sites. At least 100 adults (half male and half female) and 100 pregnant women were selected as survey subjects in each investigation site. Random urine samples were collected from all survey subjects once to detect urine iodine, creatinine and sodium contents. Venous blood samples of all pregnant women were collected to detect the serum iodine content, and the 95% medical reference range of serum iodine was established using the percentile method.Results:The medians urinary iodine, creatinine and sodium of adults in Jianshui County and Zhenxiong County were 184.7 μg/L ( n = 432), 12 355.0 μmol/L ( n = 431) and 156.5 mmol/L ( n = 420), respectively. The median urinary iodine of adults in Jianshui County was higher than that in Zhenxiong County (197.3 vs 170.2 μg/L), the difference was statistically significant ( Z = - 2.90, P = 0.003). The median serum iodine of pregnant women in Jianshui County and Zhenxiong County was 92.1 μg/L ( n = 412), with a 95% medical reference value ranged from 45.3 to 183.5 μg/L; the medians urinary iodine, creatinine and sodium of pregnant women were 138.6 μg/L ( n = 413), 12 173.0 μmol/L ( n = 408) and 152.2 mmol/L ( n = 409), respectively. The urinary iodine (154.1 vs 115.8 μg/L), urinary creatinine (13 216.0 vs 11 376.0 μmol/L) and urinary sodium (161.8 vs 141.8 mmol/L) of pregnant women in Jianshui County were higher than those in Zhenxiong County, with statistical differences ( Z = - 5.12, - 2.29, - 4.30, P < 0.05). Conclusion:Adults in Jianshui County and Zhenxiong County are at an appropriate level of iodine, but pregnant women in Zhenxiong County are at an iodine deficiency level.