HyBeacon probes are single-stranded oligonucleotides with one or more internal base(s) labeled with a fluorescent dye. When a probe forms a duplex with its target sequence, the level of fluorescence emission increases considerably. HyBeacons have been developed as new tools for rapid sequence detection and discrimination and have been employed in a wide variety of applications including infectious diagnostics and analysis of human polymorphisms. Single-labeled (FVG1) and dual-labeled (FVG11) probes were designed to analyze the factor V Leiden (R506Q) polymorphism which causes an increased risk of deep vein thrombosis and pulmonary embolism. Detection and identification of factor V alleles is performed by melting curve analysis and determination of probe melting temperature (T(m)). HyBeacon hybridization to the glutamine allele (Q) causes the formation of mismatched DNA duplexes that are detected through decreases in T(m). HyBeacon probes are included in homogeneous PCR assays to genotype samples with respect to the factor V polymorphism within 20 min, using purified DNAs and unpurified saliva/blood samples. This paper describes the preparation of homogeneous PCR assays, LightCycler target amplification, and subsequent melting curve analysis. This chapter also describes the use of homologous oligonucleotides and melting curve analysis as a method for probe evaluation.