Random mutagenesis of DNA sequences has the advantage of generating DNA sequences with novel properties, either directly in the case of aptamers or through subsequent transcription/translation of the mutated sequence in the case of proteins. In both cases no prior structural or mechanistic knowledge of the molecule is required. For sequences greater than 100 bp, one of the easiest methods to introduce the mutations is to use Error-prone PCR (EP-PCR) as discussed in this chapter. When coupled with an appropriate selection or high throughput screening methodology, PCR-based random mutagenesis can provide a powerful tool for modern molecular biologists.