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Objectives Since the onset of the COVID-19 pandemic, cases of reinfection with the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have been reported, raising additional public health concerns. SARS-CoV-2 reinfection was assessed in healthcare workers in Tunisia, as they are at the greatest exposure to infection by different variants. Methods We conducted whole-genome sequencing of the viral RNA from clinical specimens at the initial infection and suspected second infection from four healthcare workers, working at the Habib Bourguiba University Hospital (Sfax, Tunisia), who were retested positive for SARS-CoV-2 by RT-PCR after recovery from a first infection. A total of 8 viral RNAs from the patients’ respiratory specimens were obtained, which allowed us to characterize the differences between viral genomes from initial infection and positive retest. The serology status for total Ig, IgG, and IgM against SARS-CoV-2 was also determined and followed after the first infection. Results We confirmed by whole-genome sequencing of the viral samples that all four cases experienced a reinfection event. The interval between the two infection events ranged from 45 to 141 days and symptoms were milder in the second infection for two patients and more severe for the two remaining cases. Reinfection occurred for all four cases, despite the presence of antibodies for three of them. Conclusion This study adds to the rapidly growing evidence of COVID-19 reinfection, where viral sequences were used to confirm infection by distinct isolates of SARS-CoV-2 in healthcare workers. These findings suggest that individuals, who are exposed to different SARS-CoV-2 variants, might not acquire sufficiently protective immunity through natural infection and emphasis the necessity of their vaccination and the regular follow-up of their immune status both in quantitative and qualitative terms.