Abstract: The Hatschek's pit is an important component of the reproductive endocrine regulation axis in amphioxus. To further elucidate the relationship between the morphological characteristics of the Hatschek's pit and gonadal development in amphioxus, and to enrich the biological information of amphioxus, we used histological sectioning and HE staining techniques to observe the morphological changes of the Hatschek's pit in Florida amphioxus at different stages of gonadal development. The results showed that at the beginning and early stages of the gonads in Branchiostoma floridae, Hatschek's pit was located near the notochord of the amphioxus, with a thin film separating it, forming an approximately symmetrical inverted “concave” shape; During the development and maturation of the gonads, Hatschek‘s pit has a thickened top, clear cytoplasm, enlarged on both sides towards the nest, smaller gaps within the nest, and a shorter and wider shape; During the period of gonadal emptying and degeneration, the two sides of the Hatschek's pit are elongated and the space inside the pit is narrow. This study elucidates the developmental process of Hatschek‘s pit in Branchiostoma floridae and provides preliminary scientific references for the industrial research of amphioxus.