*Purpose: Resistant Cytomegalovirus (CMV) infection has emerged to cause significant impact on morbidity and mortality after solid organ transplantation (SOT). There is limited data on viral subpopulations during the course of CMV infection. Using Next-Generation Sequencing (NGS), we aimed to define the CMV subpopulations at onset and during the course of CMV infection

*Methods: After IRB approval, stored clinical samples from 18 patients with CMV viremia were tested by NGS. We performed NGS on a total of 44 frozen serum samples. All first quantifiable samples for all 18 patients were tested. 4 patients developed protracted course of CMV viremia and all their available samples were tested by NGS to detect the kinetics of UL97/UL54 mutant subpopulations.

*Results: No UL97/UL54-mutation associated with resistance to CMV DNA polymerase was detected at onset of CMV infection (in antiviral-naïve or experienced patients). Four of 18 patients developed protracted CMV viremia, associated with UL97/54 resistance mutations. Patient 1 had UL97 A594V mutation (5-10 fold ganciclovir resistance). Patient 2 had changing viral subpopulations at different time points during infection. Those mutations were not known at the time of clinical treatment and patient resolved the infection without any targeted therapeutic modifications. Patient 3 has ganciclovir refractory CMV infection, but Sanger sequencing did not identify any resistance mutations. However, NGS found a subpopulation with UL54 E756K (associated with ganciclovir and cidofovir resistance); however, this mutation was no longer detected on subsequent specimens. Patient 4 developed breakthrough low level CMV viremia while receiving secondary valganciclovir prophylaxis. Sanger sequencing did not detect resistance mutations, but NGS revealed predominance of UL97 H520Q (associated with 5-10 fold ganciclovir resistance) mutation at end of prolonged IV ganciclovir treatment.

*Conclusions: UL97/UL54 resistance mutations are not detected at onset of CMV viremia. In our limited sample set, NGS may detect UL97/UL54 mutations that would otherwise not be detected by traditional Sanger sequencing.

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