Recent studies have reported several larvae of an unidentified Amblyomma species on passerine birds in Atlantic rainforest fragments in southeastern Brazil. These larvae yielded a unique 16S rRNA haplotype designated as Amblyomma sp. haplotype Nazaré, which showed nucleotide identity levels of 91% to Amblyomma parkeri Fonseca & Aragão, 1952 and 88% to Amblyomma longirostre (Koch, 1844). Herein, we describe Amblyomma sp. haplotype Nazaré as a new species, Amblyomma romarioi n. sp. Martins, Luz & Labruna, through a formal description of the male and female adult stages. Amblyomma romarioi is morphologically and genetically most closely related to A. parkeri, A. longirostre and Amblyomma geayi Neumann, 1899. Among males, the rectangular basis capituli and rounded coxa I spurs separates A. romarioi from A. parkeri, A. longirostre, and A. geayi, which have basis capituli triangular or slightly hexagonal, and pointed coxa I spurs. Among females, the V-shaped genital aperture and coxa I rounded spurs of A. romarioi contrasts to the U-shaped genital aperture and coxa I pointed spurs in A. parkeri, A. longirostre, and A. geayi. Larvae of A. romarioi have been collected on 24 species of passerines. The few records of nymphs and adults were on the black-fronted titi monkey Callicebus nigrifrons (Spix, 1823). The current distribution of A. romarioi is restricted to the Brazilian Atlantic rainforest, southeastern Brazil, in areas with altitude between 363 and 1600 m, within the distribution of C. nigrifrons. We discuss ecological features of Amblyomma romarioi, comparatively to A. parkeri, A. longirostre and A. geayi. The present study increases the Brazilian tick fauna to 74 species.