Thyroid cancer incidence is higher in World Trade Center (WTC) responders compared with the general population. It is unclear whether this excess in thyroid cancer is associated with WTC-related exposures or if instead there is an over-diagnosis of malignant thyroid cancer among WTC first responders due to enhanced surveillance and physician bias. To maximize diagnostic yield and determine the false positive rate for malignancy, the histological diagnoses of thyroid cancer tumors from WTC responders and age, gender, and histology matched non-WTC thyroid cancer cases were evaluated using biomarkers of malignancy. Using a highly accurate panel of four biomarkers that are able to distinguish benign from malignant thyroid cancer, our results suggest that over-diagnosis by virtue of misdiagnosis of a benign tumor as malignant does not explain the increased incidence of thyroid cancer observed in WTC responders. Therefore, rather than over-diagnosis due to physician bias, the yearly screening visits by the World Trade Center Health Program are identifying true cases of thyroid cancer. Continuing regular screening of this cohort is thus warranted.