In 2000, we initiated an investigation on the tick fauna of Rondônia State, where we collected many specimens of Amblyomma scalpturatum Neumann, 1906 and Amblyomma incisum Neumann, 1906. In addition, we also collected a third group of ticks that were morphologically closely related to those 2 species, but sufficiently different to be considered a distinct species; members of this group were subsequently identified as Amblyomma latepunctatum Tonelli-Rondelli, 1939, through comparison with the type specimens of this taxon. Herein, we redescribe both sexes of A. scalpturatum and A. incisum, the female of A. latepunctatum, and provide the first description of the male of this latter species. Molecular analysis of the second internal transcribed spacer (ITS2) from rDNA of specimens of the 3 species supports morphological results. Examination of both A. scalpturatum and A. incisum deposited in different tick collections revealed that A. latepunctatum appeared relatively frequently in the vials believed to contain specimens of A. incisum or A. scalpturatum. Before this study, A. latepunctatum was considered a synonym of A. scalpturatum. Herein, we provide morphological and molecular evidence to validate the species A. latepunctatum. The South American tapir (Tapirus terrestris) seems to be the primary host for the adult stage of A. latepunctatum, A. scalpturatum, and A. incisum.