Anti-cytomegalovirus (CMV) prophylaxis is recommended in D+R− kidney transplant recipients (KTR), but is associated with a theoretical increased risk of developing anti-CMV drug resistance. This hypothesis was retested in this study by comparing 32 D+R− KTR who received 3 months prophylaxis (valganciclovir) with 80 D+R− KTR who received preemptive treatment. The incidence of CMV infections was higher in the preemptive group than in the prophylactic group (60% vs. 34%, respectively; p = 0.02). Treatment failure (i.e. a positive DNAemia 8 weeks after the initiation of anti-CMV treatment) was more frequent in the preemptive group (31% vs. 3% in the prophylactic group; p = 0.001). Similarly, anti-CMV drug resistance (UL97 or UL54 mutations) was also more frequent in the preemptive group (16% vs. 3% in the prophylactic group; p = 0.05). Antiviral treatment failures were associated with anti-CMV drug resistance (p = 0.0001). Patients with a CMV load over 5.25 log10 copies/mL displayed the highest risk of developing anti-CMV drug resistance (OR = 16.91, p = 0.0008). Finally, the 1-year estimated glomerular filtration rate was reduced in patients with anti-CMV drug resistance (p = 0.02). In summary, preemptive therapy in D+R− KTR with high CMV loads and antiviral treatment failure was associated with a high incidence of anti-CMV drug resistance.