Abstract “Fish-out-of-water” offer ecologists and evolutionary biologists ideal opportunities to study the evolution of stress resistance in vertebrate species. Annual killifishes (Cyprinodontiformes: Aplocheiloidei) constitute one of the most intriguing systems to study fish-out-of-water. “Annual” fishes possess a suite of complex and key features that include diapause stages and desiccation resistance, allowing them to complete their life cycle in seasonal bodies of water. Embryos of some non-annual killifishes have been shown to exhibit pre-hatching delays or symptoms of dormancy (similar to diapause), therefore, these species may represent an intermediate phenotype in the evolution of an annual lifestyle. The non-annual killifish Aplocheilus lineatus undergoes such hatching delay during aerial incubation. We use this species to study gene expression differences among water-incubated and aerially-incubated embryos. Differentially expressed genes are characterized and compared with expression patterns during diapause in annual species and with other developmental stages in non-annual killifishes. The annotation of 560 differentially expressed transcripts provides insight into how delayed-hatching embryos of Aplocheilus lineatus react to aerial incubation and suggest that delayed hatching is a phenomenon distinct from the diapause stages of related annual species. Similar patterns of gene expression are shared among Aplocheilus and other egg stranding and amphibious fishes.