Background: Increased public attention toward health and quality-of-life issues has led to more intensified screening for various medical conditions, including hypothyroidism. A falling serum thyrotropin (s-TSH) at initiation of levothyroxine (LT4) treatment has been reported in the United Kingdom between 2001 and 2009, indicating a falling TSH threshold, which may lead to less benefit from therapy and possibly overtreatment. The aim of this study was to investigate changes in s-TSH threshold used by general practitioners to initiate LT4 therapy between 2001 and 2015 in Copenhagen. Methods: Retrospective analysis was conducted of all s-TSH measurements between 2001 and 2015 performed at the general practitioners' joint laboratory merged with The Danish Register of Medicinal Products Statistics and The Danish National Patient Registry. For each year, both the median s-TSH at therapy initiation and the estimated treatment threshold were calculated from all s-TSH measurements performed in that year, representing the s-TSH level where the estimated probability of starting LT4 therapy was 50%. Results: A total of 929,684 individuals with 2,975,277?s-TSH measurements were included in the calculations. The size and composition of the study population remained virtually unchanged. During the study period, the number of performed s-TSH measurements increased from 110,886 to 292,911 (164%), and the number of patients initiating LT4 therapy increased from 786 to 1825 (132%), though this was comparably unchanged from 2010 to 2015. The median s-TSH at therapy initiation decreased from 10 mIU/L (interquartile range 5.2-29.7 mIU/L) in 2001 to 6.8 mIU/L (interquartile range 5.1-11 mIU/L) in 2015, while the estimated treatment threshold decreased from 28.3 mIU/L [confidence interval 21.0-40.2 mIU/L] in 2001 to 14.2 mIU/L [confidence interval 12.0-18.0 mIU/L] in 2007. In 2015, 25% of patients started LT4 therapy with s-TSH ?5 mIU/L, and during the entire period, 50% of patients started therapy with a single s-TSH measurement >5 mIU/L. Conclusions: This study performed on a sizeable primary care population demonstrates a considerable fall in the threshold for initiating LT4 therapy in hypothyroid patients. This increases the risk of futile treatment as well as overtreatment.