Recent studies suggest that killer cell immunoglobulin-like receptors (KIR) genotyping may be relevant to bone marrow/stem cell transplantation. The objective of this research was to develop a bead-based reverse sequence-specific oligonucleotide DNA hybridization assay for KIR genotyping using the Luminex® platform. The oligonucleotide probes were designed to recognize 14 currently known KIR genes and two pseudogenes, as well as null alleles. A unique probe design was used to allow detection of two cis-polymorphic regions for typing of KIR2DS4, because the functioning alleles cannot readily be assigned by conventional probe detection systems. Assay performance was validated using DNA samples previously typed by the 13th International Histocompatibility Working Group with 100% concordant results.