Information on the altitudinal distribution of the hard ticks Amblyomma aureolatum and Amblyomma ovale in Brazil is scarce and mainly limited to occasional records. In this study we report our evaluation of records on the altitudinal distribution of A. aureolatum and A. ovale collected from dogs and humans and directly from the environment (hostquesting ticks), based on active or passive procedures. The collections were conducted in rural areas of municipalities in the state of Rio de Janeiro, Brazil between 2013 and 2017. Active procedures consisted of dragging or flagging, visual examination of vegetation and removal of ticks present on the authors’ clothing or on infested dogs. Overall, 222 ticks were collected. The altitudes at the collection sites ranged from 98 to 1220 m a.s.l. We noted a significant difference in the altitudinal distribution of A. aureolatum and A. ovale (Mann–Whitney U-test, U = 518.5, P < 0.001). The overlap of these two species occurred at altitudes of between 650 and 900 m a.s.l. The results indicated that the higher the altitude, the greater the probability for the occurrence of A. aureolatum and, conversely, the lower the likelihood for the occurrence of A. ovale. The findings of this study improve currrent knowledge on the bioecology of these tick species and have implications for studies on the epidemiology of spotted fever in Brazil.