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Purpose: To compare the impact of COVID-19 pandemic on 2-[18F]FDG PET/CT imaging work-flow during the three waves in a medical institution of southern of Italy. Methods: We retrospectively reviewed the numbers and results of 2-[18F]FDG PET/CT studies acquired during the following three periods of the COVID-19 waves: 1) February 3-April 30, 2020; 2) October 15, 2020–January 15, 2021; and 3) January 18-April 16, 2021. Results: A total of 861 PET/CT studies in 725 patients (388 men, mean age 64 ± 4 years) was acquired during the three waves of COVID-19 pandemic. The majority (94%) was performed for diagnosis/staging (n = 300) or follow-up (n = 512) of neoplastic diseases. The remaining 49 studies (6%) were acquired for non-oncological patients. The distribution of number and type of clinical indications for PET/CT studies in the three waves were comparable (p = 0.06). Conversely, the occurrence of patients positive for COVID-19 infection progressively increased (p < 0.0001) from the first to third wave; in particular, patients with COVID-19 had active infection before PET/CT study as confirmed by molecular oro/nasopharyngeal swab. Conclusion: Despite the restrictive medical measures for the emergency, the number of 2-[18F]FDG PET/CT studies was unchanged during the three waves guaranteeing the diagnostic performance of PET/CT imaging for oncological patients.