A new species of Ami Pérez-Miles, 2008 is described from the state of Amazonas in Brazil based on three males from Manaus. Ami valentinae sp. nov. is closely related to A. armihuariensis and A. caxiuana by the presence of a granular area on the embolus, but it differs from the first species by the presence of two retrolateral process on the male palpal tibia and differs by the latter by the morphology of the male palpal organ. A. valentinae sp. nov. is the first species of Ami reported for the Amazonas state and second species described for Brazil.