Abstract Background We report spike protein-based lineage and AZD7442 (tixagevimab/cilgavimab) neutralizing activity of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants identified from breakthrough infections in the PROVENT preexposure prophylaxis trial. Methods Variants identified from PROVENT participants with reverse-transcription polymerase chain reaction-positive symptomatic illness were phenotypically assessed to determine neutralization susceptibility of variant-specific pseudotyped virus-like particles. Results At completion of 6 months' follow-up, no AZD7442-resistant variants were observed in breakthrough coronavirus disease 2019 (COVID-19) cases. SARS-CoV-2 neutralizing antibody titers were similar in breakthrough and nonbreakthrough cases. Conclusions Symptomatic COVID-19 breakthrough cases in PROVENT were not due to resistance-associated substitutions in AZD7442 binding sites or lack of AZD7442 exposure. Clinical Trials Registration NCT04625725.