Summary Natural killer (NK) cells exert anti‐viral effects after haematopoietic stem cell transplantation (HSCT). The balance between inhibition and activation of NK cells determined by the inherited repertoire of killer cell immunoglobulin‐like receptors (KIR) genes may influence Epstein–Barr virus (EBV) reactivation after transplantation. To evaluate the relative contributions of KIR genotypes to EBV reactivation, we prospectively enrolled 300 patients with malignant haematological disease who were suitable for haploidentical HSCT. Univariate analysis showed that donors with KIR2DS1, KIR2DS3 or KIR3DS1 genes were associated with an increased risk of EBV reactivation hazard ratio (HR) 1·86, 95% confidence interval (CI) 1·19–2·9, P = 0·0067; HR 1·78, 95% CI 1·07‐2·97, P = 0·027; HR 1·86, 95% CI 1·19‐2·91, P = 0·0065 respectively. Multivariate analysis revealed that the presence of KIR2DS1, KIR2DS3 or KIR3DS1 genes was associated with increased EBV reactivation after HSCT. This effect was more evident in the absence of the cognate ligands for the corresponding activating receptors. Our present data firstly showed that donors with activating KIR genes, specifically activating KIR2DS1, KIR2DS3 and KIR3DS1, had an increased risk of EBV reactivation. Precaution for patients whose donors carry activating genes will help prevent EBV reactivation and improve patient prognosis after HSCT.