The prevalence and clinical significance of de novo 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 (n = 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) (n = 772) and the de novo TgAb-positive group in whom TgAbs became detectable (n = 40). We then assessed whether de novo appearance of TgAb was associated with DTC structural recurrence by using the Kaplan-Meier method. The de novo detection of TgAb occurred in 5% of DTC patients. Recurrence of DTC in the TgAb persistently negative group compared with the de novo TgAb-positive group did not differ significantly (9.6% vs. 15.0%, p = 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 de novo 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 de novo TgAb detection to be 5% among initially TgAb-negative patients. We did not find a statistically significant association between de novo TgAb development and DTC structural recurrence. Larger prospective studies are required to confirm these findings and further assess the significance of de novo TgAb detection in the follow-up of DTC.