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Leptopanchax opalescens (Myers, 1942) is a small seasonal killifish endemic to coastal drainages in the state of Rio de Janeiro, Brazil. Leptopanchax opalescens was recaptured in temporary open vegetation pools (22°42.35’S, 43°41.59’W) in the hydrographic basin of the Guandu River, state of Rio de Janeiro, Brazil, after eight years without records. Information on habitat and population parameters (Capture per unit area (CPUA), size, sex ratio and length-weight relationship (LWR)) are given to provide subsidies for the conservation of this species. Specimens were captured during the rainy season (January-April 2020) in shallow pools (mean depth = 21 ± 12 s.d. cm), with relatively acidic pH (5.2 ± 0.8) and low concentrations of dissolved oxygen (3.5 ± 1 mg/L). The estimated CPUA of the population was 0.89 fish/m2. The total length (TL) of males ranged from 21.1 to 28.8 cm, and females ranged from 17.0 to 31.2 mm. Males were significantly larger (t-test = 2.04, p < 0.05) than females, which were in greater proportion (2.83Female: 1Male, p < 0.001). A positive allometric coefficient (b = 3.19) was found through length-weight relationship curve, indicating that individuals of the species gain proportionally more increment in weight than in length. Leptopanchax opalescens is a critically endangered species, and the results of this study contribute to the knowledge of the distribution, population structure and conservation of the species and its natural habitats.