Background: This study was conducted to determine the persistence of SARS-CoV-2 RNA in the nasopharynx, blood, urine and stool of patients with COVID-19.Methods: In this hospital based longitudinal study, 100 confirmed COVID-19 cases were recruited, between March and August 2020 in Guilan province (Northern Iran). Nasopharynx, blood, urine and stool samples were obtained from each patient at the time of hospital admission, discharge, followed by one week after discharge and every 2 weeks until all samples were negative for SARS-CoV-2 RNA by reverse transcription polymerase chain reaction (RT-PCR). Survival analysis was used to identify the duration of virus persistence over time.Results: Positive blood, urine, stool RT-PCR were detected in 24%, 7% and 6% of patient respectively. The median duration of virus persistence in blood, urine and stool were 7 days (95% CI: 6.07–7.93), 6 days (95% CI: 4.16–8.41) and 13 days, 95% CI: 6.96–19.4), respectively. The maximum duration of virus persistent in blood, urine and stool were 17, 11 and 42 days from admission, respectively. Conclusions: According our results, until obtaining definite evidence of the duration of infective viral shedding, prolonged isolation duration at least 25 days from admission to hospital and strict hygienic measures for about one month were recommended.