Rickettsiales bacteria represent a major global public health threat, yet their distribution patterns along ecological gradients remain poorly characterized. The distinctive east-west biome transition in Inner Mongolia provides an ideal model for studying how environmental factors shape the prevalence and diversity of Rickettsiales. Molecular screening for Rickettsia, Anaplasma, and Ehrlichia species was performed on 2372 ticks collected from diverse hosts and environmental sources across five leagues in Inner Mongolia. Nine tick species from four genera were identified, with Dermacentor silvarum being the dominant species, accounting for 24.7% of collected specimens. The overall infection rate of Rickettsia spp. was 59.8%, encompassing seven species. Ana-plasma spp. infection was detected at 13.9%, involving five species, while Ehrlichia spp. showed a prevalence of 5.6%, comprising three species. A distinct east-west divergence in pathogen distribution was observed. Eastern forest-steppe regions exhibited higher Rickettsia prevalence at 62.5%, primarily vectored by Ixodes persulcatus. Western arid regions demonstrated elevated infection levels of Anaplasma (26.4%) and Ehrlichia (7.7%), mainly associated with Dermacentor nuttalli and Hyalomma asiaticum. Co-infections occurred in 12.3% of ticks, with Rickettsia raoultii and Anaplasma ovis co-infection peaking at 65.4% in D. nuttalli from Urad Back Banner, indicating a strong association with co-detection patterns in desert-steppe environments. Phylogenetic analyses based on 16S rRNA, gltA, and groEL genes confirmed species identification and revealed geographic genetic variations. These findings provide baseline data on tick-borne pathogens in Inner Mongolia and highlight the influence of ecological heterogeneity on pathogen distribution. Region-specific surveillance and control strategies that consider environmental conditions and dominant tick species may improve the management of tick-borne disease risks in ecologically diverse regions.