The Cerrado Biome is the second largest in Brazil covering roughly 2 million km2, with varying features throughout its area. The Biome is endangered but it is also source of animal species for rural, green urban and degraded rainforest areas. Ticks are among Cerrado species that establish at anthropogenic sites and although information about them is steadily increasing, several features are unknown. We herein report tick species, abundance and some ecological relationships within natural areas of the Cerrado at higher altitudes (800–1500 m) within and around Serra da Canastra National Park, in Minas Gerais State Brazil. In total of 1196 ticks were collected in the environment along 10 campaigns held in 3 years (2007–2009). Amblyomma sculptum was the most numerous species followed by Amblyomma dubitatum and Amblyomma brasiliense. Distribution of these species was very uneven and an established population of A. brasiliense in the Cerrado is reported for the first time. Other tick species (Amblyomma ovale, Amblyomma nodosum, Amblyomma parvum, Ixodes schulzei and Haemaphysalis leporispalustris) were found in lesser numbers. Domestic animals displayed tick infestations of both rural and urban origin as well as from natural areas (Rhipicephalus sanguineus sensu lato, Rhipicephalus microplus, Dermacentor nitens, A. sculptum, A. ovale, Amblyomma tigrinum, Argas miniatus). Amblyomma sculptum had the widest domestic host spectrum among all tick species. DNA of only one Rickettsia species, R. bellii, was found in an A. dubitatum tick. Several biological and ecological features of ticks of the studied areas are discussed.