Τhe clinical significance of de novo post‐transplant anti‐HLA donor‐specific antibodies (DSA) was evaluated using 4241 serum samples collected between 2000 and 2007 from 597 renal transplant recipients. Patients transplanted before December 1996 (n = 77) were included in the historic group and those transplanted thereafter (n = 520) were included in the study group. All recipients were negative for DSA before transplantation (Tx). Post‐Tx, de novo DSA were detected in 92/597 (15.4%) patients, while 196 had third party anti‐HLA antibodies (DSA‐negative). DSA were more frequent in the historic group (33.8%) compared with the study group (12.7%) (P < 0.001). Anti‐HLA class‐II DSA predominated in both groups (84.6% vs. 69.7%). Recipients of HLA class II‐incompatible grafts developed DSA more frequently than those receiving HLA class II‐compatible grafts (17.9% vs.7.9%, P = 0.003), directed mainly against HLA‐DQ graft molecules (64/446, 14.4%). DSA production was not different between presensitized and nonsensitized patients (P = 0.842). Graft survival was higher in patients without antibodies compared with DSA‐positive (log‐rank test, P = 0.002) and DSA‐negative patients (log‐rank test, P = 0.002). Univariate and multivariate analysis showed independent association for DSA class I (HR = 31.78), DSA class II (HR = 20.92) and non‐DSA (HR = 5.94) and graft failure. We conclude that HLA class II incompatible graft transplantations need careful monitoring and should be avoided in high immunological risk cases.