As an indispensable essential amino acid in the human body, lysine is extremely rich in edible mushrooms. The α-aminoadipic acid (AAA) pathway is regarded as the biosynthetic pathway of lysine in higher fungal species in Agaricomycetes. However, there is no deep understanding about the molecular evolutionary relationship between lysine biosynthesis and species in Agaricomycetes. Herein, we analyzed the molecular evolution of lysine biosynthesis in Agaricomycetes. The phylogenetic relationships of 93 species in 34 families and nine orders in Agaricomycetes were constructed with six sequences of LSU, SSU, ITS (5.8 S), RPB1, RPB2, and EF1-α datasets, and then the phylogeny of enzymes involved in the AAA pathway were analyzed, especially homocitrate synthase (HCS), α-aminoadipate reductase (AAR), and saccharopine dehydrogenase (SDH). We found that the evolution of the AAA pathway of lysine biosynthesis is consistent with the evolution of species at the order level in Agaricomycetes. The conservation of primary, secondary, predicted tertiary structures, and substrate-binding sites of the enzymes of HCS, AAR, and SDH further exhibited the evolutionary conservation of lysine biosynthesis in Agaricomycetes. Our results provide a better understanding of the evolutionary conservation of the AAA pathway of lysine biosynthesis in Agaricomycetes.