Although human leukocyte antigen (HLA) mismatch is often thought to be associated with a high incidence of cytomegalovirus (CMV) reactivation, it is not clear whether this process is mediated by HLA mismatch or other factors, such as acute graft-versus-host disease (aGVHD). Here we focused on cord blood transplantation (CBT) and examined the effects of HLA mismatch on the incidence of CMV reactivation while minimizing the effects of aGVHD. In a multivariate analysis considering aGVHD as a time-dependent covariate, a significant effect on the incidence of CMV reactivation was noted for HLA disparity (hazard ratio [HR]: 0.54 for 8/8 match compared with 3-allele mismatch) and development of aGVHD (HR: 1.26). Next, in an analysis excluding cases that developed aGVHD, the incidences of CMV reactivation for 8/8 match and 1-allele mismatch were low compared with those for other mismatches. These findings were supported by the multivariate analysis (HR: 0.49 for 8/8 match and 0.64 for 1-allele mismatch compared with 3-allele mismatch). Together, these results suggested that HLA mismatch was involved in CMV reactivation and was associated with high morbidity of opportunistic infection after CBT.