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Cordyceps militaris is a famous traditional edible and medicinal fungus in Asia, and its fruiting body has rich medicinal value. The molecular mechanism of fruiting body development is still not well understood in C. militaris. In this study, phylogenetically analysis and protein domains prediction of the 14 putative chitinases were performed. The transcription level and enzyme activity of chitinase were significant increased during fruiting body development of C. militaris. Then, two chitinase genes (Chi1 and Chi4) were selected to construct gene silencing strain by RNA interference. When Chi1 and Chi4 genes were knockdown, the differentiation of the primordium was blocked, and the number of fruiting body was significantly decreased approximately by 50% compared to wild-type (WT) strain. The length of the single mature fruiting body was shortened by 27% and 38% in Chi1- and Chi4-silenced strains, respectively. In addition, the chitin content and cell wall thickness were significantly increased in Chi1- and Chi4-silenced strains. These results provide new insights into the biological functions of chitinase in fruiting body development of C. militaris. [ABSTRACT FROM AUTHOR]