In order to explore the correlation among the common genes, specific genes and differentially expressed genes (DEGs) of mycelium (AaM) and spore (AaS) samples of Ascosphaera apis cultured in lab condition with the fungal growth, development, reproduction and pathogenicity, comparative transcriptome analysis of AaM and AaS was performed using RNA-seq technology and bioinformatic methods, followed by RT-qPCR to confirm the reliability of the sequencing data. The results showed that there were 7,362 genes shared by AaM and AaS, involving 44 functional terms and 122 pathways. The specific genes of AaM and AaS were 195 and 278, respectively. In AaM vs AaS comparison group, 977 up-regulated genes and 687 down-regulated genes were discovered, which were associated with 32 and 32 functional terms, respectively. Additionally, the DEGs mentioned above were related to 113 pathways such as glycolysis/gluconeogenesis and biosynthesis of secondary metabolites. Based on comparative transcriptome analysis, it’s revealed that the common genes, specific genes and DEGs may participate in the growth, development and reproduction process of A.apis mycelium and spore through regulating several pivotal pathways such as cell activity, metabolic process, endocytosis and MAPK signaling pathway.