Hybridization-based assays for the detection of nucleic acids including in situ hybridization are increasingly being utilized in a wide variety of disciplines such as cytogenetics, microbiology, and histology. Generally in situ hybridization assays utilize either cloned genomic probes for the detection of DNA sequences or oligonucleotide probes for the detection of DNA or RNA sequences. Alternately, PNA probes are increasingly being utilized in a variety of in situ hybridization assays [1, 2]. The neutral backbone of the PNA molecule allows for the PNA probes to bind to DNA or RNA under low ionic strength conditions that will either disfavor reannealing of complimentary genomic sequences or are denaturing for RNA secondary structure but are favorable for PNA/DNA or PNA/RNA hybridization [3, 4]. For in situ hybridization assays these unique properties of PNA probes offer significant advantages that allow for the development of fast, simple, and robust assays (Figs. 14.1 and 14.2).