Cytomegalovirus (CMV) transmission via stem cells or marrow in CMV donor seropositive/recipient seronegative (D+/R−) hematopoietic cell transplantation (HCT) is surprisingly inefficient, and factors associated with transmission in these high-risk HCT recipients are unknown. In a retrospective cohort of D+/R− HCT recipients, cumulative incidence curve estimates were used to determine posttransplantation rates of CMV and multivariable Cox proportional models to assess risk factors associated with transmission. A total of 447 patients from 1995 to 2007 were eligible for enrollment. Overall, 85 of 447 (19.0%) acquired CMV at a median of 49 days (IQR 41-60) posttransplantation. CMV disease before day 100 occurred in 6 of 447 (1.3%) patients and in 7 of 447 (1.6%) after day 100. The donor graft, specifically the total nucleated cell count (adjusted hazard ratio [HR] 2.7; 95% confidence interval [CI], 1.4-4.7, P = .0002), was the only factor associated with CMV transmission in multivariable analyses. Notably, the source stem cells (marrow versus peripheral blood stem cell [PBSC]), screening method, and graft-versus-host disease (GVHD) were not associated with transmission. Thus, a highly cellular graft was the only identifiable risk factor associated with CMV transmission, suggesting that viral genomic content of the donor graft determines transmission efficiency in D+/R− HCT recipients.