Killer-cell immunoglobulin-like receptors (KIRs) on natural killer (NK) cells recognize groups of HLA class I alleles. Recent work suggests that KIR genotype may affect the outcome of hematopoietic stem-cell transplants and that prospective KIR typing maybe of benefit in future matching of donors and recipients. A simple and informative KIR genotyping method was developed using a multiplex polymerase chain reaction-sequence-specific primer strategy. This method contains four multiplex reactions for detecting all functional KIR genes, including some 2DS4 variants that harbor a common deletion. Primer pairs were designed to provide short amplicons (108-565 bp) that can be analyzed by agarose gel electrophoreses or by automated electrophoretic systems. This method was evaluated in a blinded survey with the NK/KIR Phase II QC Panel (a total of 16 cell lines) from the 14th International Histocompatibility Workshop (IHWS), and the results are 100% concordant with the consensus genotype. Results in further KIR genotyping of 20 reference cell lines from the 10th IHWS were consistent with previously published genotypes, matching those of one study in instances where different genotypes have been previously reported. The genotypes obtained in this study may be helpful to other labs developing KIR genotyping methods in resolving typing discrepancies and in detecting common deletion variants of 2DS4. This method can save labor and reagent costs. It provides good results from partially degraded template DNA due to short amplicons in this method. It is convenient to use in both clinical and research laboratories.