Detection of single-nucleotide polymorphisms (SNPs) in high-throughput studies promises to be an expanding field of molecular medicine in the near future. Highly specific, simple, and accessible methods are needed to meet the rigorous requirements of single-nucleotide detection needed in pharmacogenomic studies, linkage analysis, and the detection of pathogens. Molecular beacons present such a solution for the high-throughput screening of SNPs in homogeneous assays using the polymerase chain reaction (PCR). Molecular beacons are probes that fluoresce on hybridization to their perfectly complementary targets. In recent years they have emerged as a leading genetic analysis tool in a wide range of contexts from quantification of RNA transcripts, to probes on microarrays, to single-nucleotide polymorphism detection. The majority of these methods use PCR to obtain sufficient amounts of sample to analyze. The use of molecular beacons with other amplification schemes has been reliably demonstrated, though PCR remains the method of choice. Here we discuss and present how to design and use molecular beacons to achieve reliable SNP genotyping and allele discrimination in real-time PCR. In addition, we provide a new means of analyzing data outputs from such real-time PCR assays that compensates for differences between sample condition, assay conditions, variations in fluorescent signal, and amplification efficiency. The mechanisms by which molecular beacons are able to have extraordinary specificity are also presented.