Nanofluidic channels are used to separate DNA molecules by length and applications of the channels to other biomolecules are discussed. The problem of separating DNA and biomolecules by length, charge, or other physical characteristics has consumed a tremendous amount of time and resources over the latter half of the twentieth century and the beginning of the twenty-first century. Micro- and nanofluidic structures afford the opportunity to increase separation effectiveness while decreasing the cost of analysis. Additionally, owing to their simplicity, micro- and nanofluidic devices offer the possibility of modeling molecular motion through confining environments from fundamental physical principles. Three nanofluidic devices for separating biomolecules by length are discussed herein. A description of their physical operation is given, and results indicating their effectiveness are presented. Important concepts in molecular biology, polymer physics, and electrophoresis are presented, as well as a review of the trend toward miniaturization of traditional separation techniques. The physical effects manifest in the nanofluidic devices described herein are most easily leveraged in the microchip format, so the details of the fabrication processes used to manufacture these devices are also presented.