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Simple Summary: Ticks are hematophagous ectoparasites that spread diseases to both animals and humans through their bites. They are notorious for carrying various disease-causing agents, such as viruses, protozoa, and bacteria, which present substantial risks to both human and animal well-being. Continuous changes in the climate can impact both tick distribution and abundance. Understanding of the epidemiology of tick-borne Rickettsia hoogstraalii is limited, with gaps in its molecular detection, genetic characterization, and absence of data, especially from Pakistan. This study aimed to use molecular methods to genetically analyze Rickettsia species, particularly R. hoogstraalii, in Pakistan while also contributing new insights into the pathogen′s global epidemiology. For this purpose, ticks were collected from different hosts, including goats, sheep, and cattle, from six districts of Khyber-Pakhtunkhwa, Pakistan. This study is the first to genetically characterize R. hoogstraalii in Hyalomma anatolicum ticks globally and Haemaphysalis sulcata in Pakistan. This species was first described in 2006 in Croatia and has also been detected in different species of ticks in different countries. The pathogenicity of R. hoogstraalii in vertebrate hosts is not well understood. Encouraging additional research is essential to unveil the involvement of ticks in the transmission and persistence of R. hoogstraalii across various host species. Ticks are hematophagous ectoparasites that transmit pathogens to animals and humans. Updated knowledge regarding the global epidemiology of tick-borne Rickettsia hoogstraalii is dispersed, and its molecular detection and genetic characterization are missing in Pakistan. The current study objectives were to molecularly detect and genetically characterize Rickettsia species, especially R. hoogstraalii, in hard ticks infesting livestock in Pakistan, and to provide updated knowledge regarding their global epidemiology. Ticks were collected from livestock, including goats, sheep, and cattle, in six districts of Khyber Pakhtunkhwa (KP) Pakistan. Overall, 183 hosts were examined, of which 134 (73.2%), including goats (number = 39/54, 72.2%), sheep (23/40, 57.5%), and cattle (71/89, 80%) were infested by 823 ticks. The most prevalent tick species was Rhipicephalus microplus (number = 283, 34.3%), followed by Hyalomma anatolicum (223, 27.0%), Rhipicephalus turanicus (122, 14.8%), Haemaphysalis sulcata (104, 12.6%), Haemaphysalis montgomeryi (66, 8.0%), and Haemaphysalis bispinosa (25, 3.03%). A subset of 210 ticks was selected and screened for Rickettsia spp. using PCR-based amplification and subsequent sequencing of rickettsial gltA and ompB fragments. The overall occurrence rate of R. hoogstraalii was 4.3% (number = 9/210). The DNA of Rickettsia was detected in Hy. anatolicum (3/35, 8.5%) and Ha. sulcata (6/49, 12.2%). However, no rickettsial DNA was detected in Rh. microplus (35), Rh. turanicus (35), Ha. montgomeryi (42), and Ha. bispinosa (14). The gltA and ompB fragments showed 99–100% identity with R. hoogstraalii and clustered phylogenetically with the corresponding species from Pakistan, Italy, Georgia, and China. R. hoogstraalii was genetically characterized for the first time in Pakistan and Hy. anatolicum globally. Further studies should be encouraged to determine the role of ticks in the maintenance and transmission of R. hoogstraalii in different hosts. [ABSTRACT FROM AUTHOR]