This study was purposed to investigate the killer immunoglobulin-like receptor (KIR) genotype in patients with hematological malignancies. The sequence specific primer-polymerase chain reaction (PCR-SSP) technique was performed for the amplification of six inhibitory KIR genes (KIR2DL1-2DL4, 3DL1-3DL2) and six activating KIR genes (KIR2DS1-S5, 3DS1). The methods of KIR-SSP was used to determine the KIR genotypes of 54 leukemia patients, including 14 patients with acute myeloid leukemia (AML), 16 with acute lymphoblastic leukemia (ALL), 20 with chronic myeloid leukemia (CML), 3 with myelodysplastic syndrome (MDS) and 1 with acute myeloid-lymphoblast leukemia (AMLL). 54 patients were classified as high risk group (n = 27) and standard risk group (n = 27). The expression of KIR in NK cells and T cells was detected by flow cytometry. The frequencies of activating KIR genes in standard risk group were higher than those in high risk group, especially 2DS1 (p = 0.014), or 2DS2 (p = 0.046), or 3DS1 (p = 0.027). However, the frequencies of inhibitory KIR genes in standard risk group were similar to those in high risk group (p > 0.05). The frequencies of activating KIR genes were also higher in standard risk patients with acute AML, as compared with those in high risk patients with acute AML, particularly 2DS1 (66.7% vs 29.4%, p = 0.022), 2DS2 (57.6% vs 17.6%, p = 0.013), and 2DS3 (33.3% vs 5.9%, p = 0.039). The percentages of patients in high-risk group who expressed more than two kinds of activating KIRs were lower that those in standard-risk group (p = 0.035). There was no difference in the expressions of CD158a, CD158b, and CD158e on NK cells and T cells between high-risk group and standard-risk group (p > 0.05). In conclusions, different expressions of activating KIR genes were found in patients between high-risk group and standard-risk group.