The mechanism of sex determination and differentiation in animals remains a central focus of reproductive and developmental biology research, and the regulation of sex differentiation in amphioxus remains poorly understood. Cytochrome P450 Family 19 Subfamily A member 1 () is a crucial sex differentiation gene that catalyzes the conversion of androgens into estrogens. In this study, we identified two aromatase-like genes in amphioxus: and . The is more primitive and may represent the ancestral form of in zebrafish and other vertebrates, while the is likely the result of gene duplication within amphioxus. To gain further insights into the expression level of these two , we examined their expression in different tissues and during different stages of gonad development. While the expression level of the two genes differs in tissues, both are highly expressed in the gonad primordium and are primarily localized to microsomal membrane systems. However, as development proceeds, their expression level decreases significantly. This study enhances our understanding of sex differentiation mechanisms in amphioxus and provides valuable insights into the formation and evolution of sex determination mechanisms in vertebrates.