The Polymerase Chain Reaction (PCR) with its multiple applications in molecular genetic analysis is the cornerstone of modern basic and applied biomedical research. This chapter focuses on the inverse PCR technique that has been used widely over the last two decades in genotyping and chromosome walking applications for the isolation of unknown DNA sequences upstream and downstream of a known DNA region. The method is based on the use of circularized templates and primers facing outward from the known sequence, rather than primers facing each other used in conventional PCR. As a result, the original genome sequence is rearranged, and stretches of known sequence end up flanking the unknown DNA sequence in the inverse PCR product. I also discuss the special case of using outward facing primers to isolate the intergenic region between genes clustered in tandem or inverted arrangement, since it can hugely simplify the cloning of cis-regulatory sequences in new species of interest.