To The Editor, Currently, the U.S. Centers for Disease Control and Prevention, American Academy of Pediatrics and the World Health Organization advise that women who are infected with SARS-CoV-2 may choose to breastfeed with appropriate protections to prevent transmission of the virus through respiratory droplets. However, the potential for exposure to SARS-CoV-2 through breastfeeding is currently unknown. To date, case reports on breastmilk samples from a total of 24 SARS-CoV-2-infected women have been published. Of those, viral RNA was detected in ten breastmilk samples from four women. In some but not all cases, environmental contamination as the source of the virus or retrograde flow from an infected infant could not be ruled out. We established a quantitative RT-PCR assay for SARS-CoV-2 in breastmilk with a limit of detection of 250 copies per mL and validated it by spiking breastmilk from uninfected women with known amounts of viral RNA. In addition, we established tissue culture methods to detect replication-competent SARS-CoV-2 in breastmilk. No viral RNA nor culturable virus was detected after Holder pasteurization of breastmilk samples that had been spiked with replication-competent SARS-CoV-2 (see Supplement). Between March 27 and May 6, 2020, we collected and analyzed 64 serial breastmilk samples from 18 SARS-CoV-2-infected women residing in the U.S. (see Supplement for clinical characteristics). Breastmilk samples were collected before and after women had a positive SARS-CoV-2 RT-PCR test and all but one woman had symptomatic disease (see Figure). One of the 64 breastmilk samples had detectable SARS-CoV-2 RNA by RT-PCR. The positive sample was collected on the day of symptom onset but one sample 2 days prior to symptom onset and two subsequent samples, collected 12 and 41 days later, tested negative for viral RNA. In addition, a subset of 26 breastmilk samples from nine women were tested for the presence of replication-competent virus using our established culture methods, and all were negative including the one sample that tested positive for viral RNA by RT-PCR. Although SARS-CoV-2 RNA was detected in one milk sample from one of eighteen infected women, the viral culture for that sample was negative. This suggests that SARS-CoV-2 RNA does not represent replication-competent virus and that breastmilk itself is likely not a source of infection for the infant. Furthermore, when control breastmilk samples spiked with replication-competent SARS-CoV-2 virus were treated by Holder pasteurization, a process commonly performed by donor milk banks, no replication-competent virus nor viral RNA was detectable. Further research to confirm these findings is needed, as well as an examination of convalescent milk for the presence of antibodies against SARS-CoV-2.