Cytomegalovirus (CMV)-specific cellular immunity is essential in controlling CMV infection after transplantation. We investigated whether CMV-specific T cell levels predict CMV DNAemia after kidney transplantation. Using cytokine-flow cytometry, we enumerated interferon-gamma producing CMV-specific CD4+ and CD8+ T cells at serial time points among CMV-mismatched (D+/R-) and seropositive (R+) kidney recipients who received 3 months of valganciclovir prophylaxis. Among 44 patients, eight (18%) developed CMV DNAemia at a mean (+ or - SD) time of 151 (+ or - 33) days after transplantation, including two (5%) with CMV syndrome and three (7%) with tissue-invasive CMV disease. Cox proportional hazards regression analysis showed that CMV mismatch (D+/R-) status (HR: 13, 95% CI: 1.6-106.4; P = 0.02) and diabetes mellitus (HR: 5.6; 95%CI: 1.1-27.9; P = 0.03) were significantly associated with CMV DNAemia. In contrast, the percentage or change-over-time in CMV-specific CD4+ [pp65 (P = 0.45), or CMV lysate (P = 0.22)] and CD8+ [pp65 (P = 0.43), or IE-1 (P = 0.37)] T cells were not significantly associated with CMV DNAemia. CMV-specific T cell assays have limited clinical utility among CMV R+ kidney recipients who received valganciclovir prophylaxis. On the other hand, the clinical utility of CMV-specific T cell assays will need to be assessed in a larger cohort of CMV D+/R- kidney recipients who remain at high-risk of delayed-onset CMV disease.