The prevalence and clinical significance of detection of anti-thyroglobulin antibodies (TgAbs) during the follow-up of patients with differentiated thyroid cancer (DTC) is unknown. We utilized the National Thyroid Cancer Treatment Cooperative Study registry (1987-2012). Patients registered after 1996 ( = 3318) were analyzed. We identified 1545 subjects who had available TgAb status (TgAb cohort) between years 1996 and 2012, of whom 1325 were TgAb negative at first postoperative follow-up testing. From this initial TgAb-negative group, we excluded 513 patients: 423 patients who had less than 3 years of follow-up and/or fewer than three follow-up visits, 86 patients with persistent disease after initial treatment, and 4 patients with data entry errors. The remaining 812 patients were included for analysis, comprising the TgAb persistently negative group (defined as TgAb negative for at least 3 consecutive follow-up visits and at least 3 years of follow-up) ( = 772) and the TgAb-positive group in whom TgAbs became detectable ( = 40). We then assessed whether appearance of TgAb was associated with DTC structural recurrence by using the Kaplan-Meier method. The detection of TgAb occurred in 5% of DTC patients. Recurrence of DTC in the TgAb persistently negative group compared with the TgAb-positive group did not differ significantly (9.6% vs. 15.0%,  = 0.23). Baseline characteristics, histology, history of radiation exposure, staging, and median duration of follow-up were similar between the two groups. Interestingly, in all six patients who suffered a recurrence in the TgAb-positive group, the TgAbs were negative at the time of recurrence detection and became positive at a median of 2.1 (0.7-8.7) years after the structural recurrence. Utilizing a large North American DTC registry, we found the prevalence of TgAb detection to be 5% among initially TgAb-negative patients. We did not find a statistically significant association between TgAb development and DTC structural recurrence. Larger prospective studies are required to confirm these findings and further assess the significance of TgAb detection in the follow-up of DTC.