Animals develop as embryos inside eggs and must hatch to allow the organism’s free-living stage to begin. Aquatic vertebrate embryos typically hatch using their hatching gland. Hatching gland cells produce hatching enzymes that digest the chorion, the egg envelope protecting embryos from the environment. Killifish are a group of teleost freshwater fishes in the Cyprinodontiformes order. Annual killifishes inhabit seasonal freshwater pools in tropical region that periodically dry up so the adult population dies. Before the dry season, killifish spawn eggs in soil where embryos tolerate long periods of desiccation. Embryos enter different phases of arrested development, or diapauses, and require environmental cues like flooding to trigger hatching from final diapause stage. In teleost fishes, hatching enzyme genes have duplicated and sub-functionalized, resulting typically in two types, high choriolytic-like enzyme (hce), and low choriolytic-like enzyme (lce) genes. Both genes are highly dynamic with extensive copy number variation between species. Hatching enzyme repertoire and hatching gland location has thus far been unknown in annual killifishes. We infer the evolutionary history of killifish hatching enzyme genes in multiple species and investigate hatching gland morphology of annual Rio pearlfish (Nematolebias whitei), our central killifish model species. We show lce genes are expressed in the pharyngeal and buccal cavities, consistent with closely related species. Also, we describe a common trend of increased number of hce genes in killifish compared to lce genes. We aim to better understand how environmental cues induce hatching by studying and comparing spatiotemporal expression of hatching enzyme genes in killifish to other aquatic vertebrates and their evolutionary history.