Cordyceps sinensis (Berk.) Sacc., also known as caterpillar fungus or Dong Chong Xia Cao in Chinese, is a complex consisting of the fruiting body (stroma) of the fungus and the dead caterpillar (sclerotium). Many scientists worked on cultured Cordyceps due to its rarity. Up to date, more than 37 genera fungi have been isolated from natural C. sinensis, and some mycelia products are sold as medicines or health food. Therefore, it is necessary to know whether different fungal strains produce similar chemical compositions. However, cultivation of C. sinensis has not been successfully achieved. In this study, random amplified polymorphic DNA (RAPD) assay was performed to evaluate the genetic relationships of 16 fungal strains isolated from natural C. sinensis, and five primers were available for discrimination of the investigated samples. Nucleosides among cultured mycelia of different fungal strains isolated from natural C. sinensis were similar, but they were different from natural one. Two-dimensional fluorescence difference gel electrophoresis (2D-DIGE) coupled with matrix-assisted laser desorption/ionization tandem time of flight (MALDI-TOF/TOF) mass spectrometry was carried out to systematically analyze differential expression proteins from natural and cultured Cordyceps. The results showed that 62 differentially expressed proteins between fruiting bodies and sclerotia of natural C. sinensis, 115 differential expression proteins between fruiting bodies and corresponding mycelia of Cordyceps, and 40 differentially expressed proteins between early and late stage of fruiting bodies of C. militaris were firstly identified. In addition, 72 differentially expressed proteins between fruiting bodies of natural C. sinensis and mycelia of QHFB, a novel fungus isolated from natural Cordyceps sinensis, and 111 proteins between mycelia of Hirsutella sinensis and QHFB were identified. Seven proteins are important to develop fruiting body of C. sinensis. iv Especially, enolase was first proposed to be crucial in fruiting body development of macrofungus. Besides, the cAMP signal pathway was presumed to involve in the fruiting body development of C. sinensis, meanwhile the glycometabolism, protein metabolism, energy metabolism, cell reconstruction were more active during fruiting body development. A map of metabolic pathways for fruiting body development of C. sinensis was proposed.