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Gamasellodes lavafesii Castro, Azevedo & Castilho sp. nov. is described based on the morphology of adult females collected from litter and soil in a transition region between Cerrado and Amazon Rainforest of Mato Grosso state and in the Caatinga biome of Alagoas state, respectively in midwestern and northeastern Brazil. A key for the separation of females of the 26 recognisable world species of Gamasellodes is provided.