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 (CYP19A1) 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: cyp19-like1 and cyp19-like2. Cyp19-like1 is more primitive and may represent the ancestral form of cyp19 in zebrafish and other vertebrates. Cyp19-like2, on the other hand, is likely the result of gene duplication within am-phioxus suggesting its potential role as a key gene in sex differentiation. To gain further insights into the expression patterns of these two aromatase-like, we examined their expression in different gonad tissues and during different stages of gonad development. While the expression patterns of the two genes differ in gonad tissues, both are highly expressed in the gonad primordium and are primarily localized to microsomal membrane systems. However, as development proceeds, their expression levels decrease significantly. This study enhances our understanding of sex dif-ferentiation mechanisms in amphioxus and provides valuable insights into the formation and evolution of sex determination mechanisms in vertebrates.