【目的】 冬虫夏草Ophiocordyceps sinensis是一种重要的食药用真菌，仅分布在青藏高原及其周边高海拔地区，具有独特的生存环境、生物学特性以及显著扩张的基因组。本研究旨在确定冬虫夏草菌的染色体数量，为该物种的基因组测序、进化、遗传和分子生物学研究奠定基础，也为其他虫草菌染色体计数研究提供参考。【方法】 二、三代测序辅以Hi-C组装，结合显微观察研究冬虫夏草的染色体数量。选择已组装获得基因组完成图的冬虫夏草1229菌株，首先利用昆虫细胞培养液培养获得芽生孢子，芽生孢子经秋水仙素和溶壁酶处理，获得细胞有丝分裂中期分散的染色体，DAPI荧光染色后使用激光共聚焦显微镜观察计数，结合基因组组装和显微观察的结果确定冬虫夏草染色体数量。【结果】 二、三代测序辅以Hi-C组装，得到6条包含两端端粒的完整染色体，染色体之间显示出较强的端粒互作；显微观察结果也支持冬虫夏草1229菌株的染色体数量为6条。【结论】 冬虫夏草菌的基因组包含6条染色体，芽生孢子是观察冬虫夏草菌染色体的理想材料。

[Objectives]  To investigate the chromosome number of Ophiocordyceps sinensis, an important edible and medicinal fungi endemic to Tibetan Plateau and surrounding areas of high elevation, and thereby lay a foundation for genome sequencing and other research on the evolution, genetics and molecular biology of this species, as well as help determine the chromosome number of other entomogenous fungi. [Methods]  A genome assembly using second and third generation sequencing technology supplemented with Hi-C data, in addition to microscopic observation, was used to determine the chromosome number of O. sinensis. Strain 1229, the genome of which has been sequenced and assembled to a complete level, was cultured in insect cell culture medium to acquire blastospores. Blastospores were co-incubated with colchicines, then treated with lywallzyme to obtain dispersed metaphase chromosomes. Those chromosomes were stained with fluorescent DAPI, then observed and counted under a laser confocal microscope. [Results]  Based on the genome assembly obtained from second and third generation sequencing and Hi-C data, strain 1229 of O. sinensis was found to possess six chromosomes with a strong telomere interaction between chromosomes. This number was supported by microscopic observations. [Conclusion]  The genome of O. sinensis possesses six chromosomes. Blastospores are suitable for counting the chromosomes of O. sinensis.