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Abstract Notholebias minimus is an endangered annual killifish endemic to the coastal plains of the State of Rio de Janeiro, Brazil. This study aimed to present new occurrences in the Atlantic Forest biome, provide unprecedented population features (body and egg size, fecundity, sexual ratio, and length-weight relationship – LWR), and compare changes in land use and coverage between 1985 and 2021 in biotopes located inside and outside protected areas. Three new occurrence localities were found in shallow temporary wetlands with acidic pH (6.4 ± 0.2) and low concentrations of dissolved oxygen (2.0 ± 0.9 mg/L). Males and females total length ranged from 11.1 to 31 mm and 11 to 26 mm, respectively. Batch fecundity ranged from 18 to 40 oocytes (24.8 ± 8.8), corresponding to oocytes with sizes between 800–1,006 µm (905 ± 56). Males were significantly larger than females (W = 2193.5, p = 0.0067), but both sexes occurred in similar proportions (p = 0.472). LWR showed positive allometry (b = 3.18). Biotopes located within protected areas exhibited higher conservation. Our discoveries expand the knowledge about habitat and population features of N. minimus and reinforce the importance of establishing protected areas for the conservation of annual fish biotopes.