Background: Many organisms are able to survive in extreme environments by entering a state of dormancy. In dormancy, vital activities are reduced until environmental conditions are compatible with active life. Annual killifishes show a special developmental pattern characterized by a phase of dispersion-reaggregation of the blastomeres that separates epiboly from organogenesis, and the capability to enter dormancy in diapause. High tolerance to environmental stress confers annual killifish embryos the condition of extremophiles. At present, the questions of our research group are focused on the understanding of the mechanisms involved in diapause regulation through an interdisciplinary approach. As a first step, it is necessary to characterize diapauses at morphological and physiological levels and to evaluate induction cues under laboratory conditions. In this context, we characterized diapause I in two Austrolebias species. Results: Our experimental approach to induce diapause I was successful and revealed the co-existence of two diapause I phenotypes named A and B instead of one. These phenotypes showed a tendency for lower total extractable RNA content compared with active developmental stages (80–100% epiboly and early reaggregate). Conclusions: These phenotypes are alternative diapause I stages and may have ecological relevance because both were found in embryos in natural ponds. Developmental Dynamics 246:848–857, 2017. © 2017 Wiley Periodicals, Inc.