Newborn screening for congenital hypothyroidism (CH) is performed by measuring the concentration of thyroxine (T4) and/or thyroid-stimulating hormone (TSH) in dried blood spots. Unfortunately, the levels of T4 and TSH vary due to multiple factors, and therefore the false-positive rate for the test is a challenge. We analyzed screening data from 2008 to 2017 to determine the effect of seasonal changes and manufacturer kit lot changes on T4 and TSH values and on numbers of infants referred. Over a 10-year period, we screened 2.4 million infants using commercially available fluoroimmunoassays to measure T4 and TSH concentrations in dried blood spots. During colder months, daily mean T4 and TSH values were higher and referral rates and false-positive rates were higher. However, there was no significant difference between the number of confirmed CH cases. Furthermore, in rare instances, we observed differences in T4 daily mean values during the 10-year period when manufacturer kit lot changes were made. Seasonal temperature variations influence measured T4 and TSH values and consequently lower the positive predictive value for CH testing in colder months. Newborn screening (NBS) programs should be aware that manufacturer kit lot changes may also influence T4 values.