The golden–needle mushroom Flammulina filiformis is one of the most precious cultivated edible fungi in the world. Despite recent progress in the study of F. filiformis, there is still a gap in the regulation of the mitochondrial genome during browning, which poses a serious threat to the golden–needle mushroom industry. Comparative transcriptome analysis of two isonuclear alloplasmic strains showed that changes in the mitochondrial genome lead to different gene expression and key biological pathways at different stages in the two isonuclear alloplasmic strains. Furthermore, transcriptome analysis revealed that the mitochondrial genome has a significant role in the regulation of a multitude of critical metabolic pathways relating to the browning of F. filiformis fruiting bodies. Functional enrichment analysis showed that the differentially expressed genes were mainly involved in many vital processes of mitochondria, mitochondrial membrane, and multiple amino acid metabolisms of F. filiformis. Taken together, the current study highlights the crucial role of the mitochondrial genome in the growth of F. filiformis and could be beneficial to genetic breeding of elite varieties of edible fungi.