Roundup is the most popular glyphosate-based herbicide (GBH) worldwide. These formulations kill a wide range of plants. Despite that, non-target species can be jeopardized by GBH, such as the annual fish Austrolebias nigrofasciatus. This species occurs in wetlands that dries annually. Key-adaptations permit them to live in such harsh habitats, e. i. Elevated fertility, drought-tolerant diapausing embryos and elevated thermal tolerance. We aimed to evaluate acute (96?h) effects of Roundup exposure (0.36 or 3.62?mg a. e./L) in reproduction, diapause pattern and embryonic upper thermal tolerance (EUTT) of A. nigrofasciatus. For such, we evaluated the number and diameter of embryos produced by exposed fish. Also, recently fertilized embryos were exposed and its diapause pattern was evaluated. Following 15 post exposure days (PED), we evaluated the number of somite pairs and following 30, 35 and 40 PED we evaluated the proportion of pigmented embryos (PPE). Finally, the critical thermal maximum (CTMax) of exposed embryos was assessed. Results demonstrated that couples exposed to 0.36?mg a. e./L Roundup produced less but larger embryos. Similarly, embryos exposed to 3.62?mg a. e./L Roundup had a reduced PPE following 30 PED. Finally, embryos exposed to 0.32?mg a. e./L Roundup had a CTMax reduction of 2.6?°C and were more sensitive to minor increases in heating rates. These results indicate that Roundup have negative outcomes in fish reproduction, embryonic development and EUTT. This information is of particular interest to the conservation of annual fish, considering that those are key-adaptations that permit these animals to survive the harsh impositions of ephemeral wetlands.