The Kit system, including Kit ligand and its receptor Kit, has critical roles in mammalian reproduction, especially in the ovary. Unlike mammalian species, two copies of genes are present in the zebrafish genome for both ligand (kitlga and kitlgb) and receptor (kita and kitb). Phylogenetic and chromosome synteny analyses suggest that these duplicated genes were likely derived from the third round of genome duplication in vertebrate evolution. All four Kit system members were expressed in the zebrafish ovary, suggesting potential roles for the system in ovarian development and function. As the first step toward understanding the Kit system in the zebrafish ovary, especially the differential roles of different forms of ligand and receptor, the present study was undertaken to analyze the temporal expression profiles of the Kit system during folliculogenesis. All members of the Kit system exhibited significant and distinct variation in expression, particularly in the periovulatory period. During the transition from the primary growth to secondary growth, kitlga showed a dramatic increase, suggesting a role for Kit signaling in follicle recruitment. During final oocyte maturation, kitlga expression significantly decreased after a transitional surge before germinal vesicle breakdown, whereas all other members exhibited an increased expression, with kitlgb and kita reaching the peak expression levels in the ovulated eggs. Our results provide strong evidence that the Kit system is involved in regulating zebrafish ovarian function and that different isoforms of Kit and Kit ligand may have differential roles in folliculogenesis. This provides an example for neofunctionalization of duplicated genes in vertebrates.