Myelin is a multilamellar membrane structure primarily composed of lipids and myelin proteins essential for proper neuronal function. Since myelin is a target structure involved in many pathophysiological conditions such as metabolic, viral, and autoimmune diseases and genetic myelin disorders, a reliable myelin detection technique is required that is equally suitable for light- and electron-microscopic analysis. Here, we report that single myelinated fibers are specifically stained by the gold phosphate complex, Black gold, which stains myelin in the brain, spinal cord, and peripheral nerve fibers in a reliable manner. Electron-microscopic and morphometric analyses have revealed that gold particles are equally distributed in the inner, compact, and outer myelin layers. In contrast to Luxol fast blue, the gold dye stains proteinase-sensitive myelin structures, indicating its selective labeling of myelin-specific proteins. Aiming at defining the target of gold staining, we performed staining in several mouse myelin mutants. Gold complex distribution and myelin staining in MBP(-/-)/shiverer mouse mutants was comparable with that seen in wild-type mice but revealed a more clustered Black gold distribution. This gold staining method thus provides a sensitive and specific high-resolution marker for both central and peripheral myelin sheaths; it also allows the quantitative analysis of myelinated fibers at the light- and electron-microscopic level suitable for investigations of myelin and axonal disorders.