We have developed a closed-tube assay for determination of the chemokine receptor type 5 (CCR5) 32-bp deletion allele, which protects against infections with HIV and modulates susceptibility to a variety of inflammatory diseases. This assay utilizes dissociation analysis of amplified products in the presence of Sybr Green I for allele discrimination. After having established robust conditions for the assay, we used it to genotype 590 unknown DNA samples. A blinded comparison with a procedure based upon agarose gel electrophoresis of amplified material revealed complete concordance between the two procedures. Our closed-tube assay is inexpensive and easy to carry out. Furthermore, it reduces or eliminates the risk of carry-over contamination with previously amplified products. The insights gained in this study can be applied to develop assays for genotyping of other insertion/deletion polymorphisms based upon differences in T(m) of allele-specific amplicons.