Dip-pen nanolithography (DPN) is a nanofabrication technique that can be used to directly write molecular patterns on substrates with high resolution and registration. Over the past two decades, DPN has evolved in its ability to transport molecular and material "inks" (e.g., alkanethiols, biological molecules like DNA, viruses, and proteins, polymers, and nanoparticles) to many surfaces in a high-throughput fashion, enabling the synthesis and study of complex chemical and biological structures. In addition, DPN has laid the foundation for a series of related scanning probe methodologies, for example, polymer pen lithography (PPL), scanning probe block copolymer lithography (SPBCL), and beam-pen lithography (BPL), which do not rely on cantilever tips. Structures prepared with these methodologies have been used to understand the consequences of miniaturization and open a door to new capabilities in catalysis, optics, biomedicine, and chemical synthesis, where, in sum, a process originally intended to compete with tools used by the semiconductor industry for rapid prototyping has transcended that application to advanced materials discovery. This review outlines the major DPN advances, the subsequent methods based on the technique, and the opportunities for future fundamental and technological exploration. Most importantly, it commemorates the 20th anniversary of the discovery of DPN.