Simple SummaryTicks are blood-feeding ectoparasites that transmit life-threatening pathogens to humans and animals. Only 10% of all identified tick species have been screened for different tick-borne pathogens.Hyalommaticks are associated with a wide range of pathogens includingTheileriaspecies,Babesiaspecies,Anaplasmaspecies,Ehrlichiaspecies, andRickettsiaspecies. Moreover, ticks of genusHyalommaare vectors for the Crimean-Congo hemorrhagic fever (CCHF), a serious threat endemic in Pakistan. In Pakistan, different tick species have been found positive for rickettsial agents; however,Hyalomma kumariticks have never been investigated for any potential pathogens. In this work,H. kumariticks were collected from goats and sheep, and morphologically and molecularly identified using different genetic markers. The identified ticks were screened for rickettsial agents using genetic markers that resulted in the detection ofRickettsia conoriifor the first time in this tick. A proper surveillance program should be designed to effectively avoid any zoonotic consequences associated with these rickettsial pathogens.AbstractAs a vector of wide range of pathogenic agents, ticks pose health threats to wild and domestic animals, and humans. Information is unavailable about the prevalence and spatial survey ofHyalomma kumariticks and associatedRickettsiaspp. in Pakistan. Concerning this knowledge gap, the present study aimed to molecularly detectRickettsiaspecies associated withH. kumariinfesting small ruminants in Khyber Pakhtunkhwa (KP), Pakistan. A total of 409H. kumariticks were collected from 163/295 infested hosts with an infestation rate of 55.25%. A total of 204 females, 158 males, and 47 nymphs were collected. Goats were heavily infested by 224 ticks having an infestation rate of 58.33% (98/168), whereas sheep were infested by 185 ticks having a lesser infestation rate of 51.18% (65/127). Genomic DNA extracted from ticks was used for the amplification of tick (cox I, 16S rRNA, ITS-2) species andRickettsia(gltA,ompA, andompB) partial genes. Eighty-three ticks were subjected to PCR, and 8/83 (9.6%) were found positive for rickettsial agents. Thecox Iand 16S rRNA sequences ofH. kumarishowed 98.90–99.74% identity withH. kumarisequences reported from Pakistan, and phylogenetically clustered to the corresponding species reported from Pakistan and India. The obtained rickettsialgltA,ompA, andompBsequences showed 100% identity withRickettsiasp. of theRickettsia conoriireported from Pakistan. In the phylogenetic trees, rickettsial sequences clustered with uncharacterizedRickettsiasp. from Pakistan andR. conoriifrom Israel, Russia, South Africa, and India. The present molecular based detection ofH. kumari-associatedR. conoriiwill facilitate effective surveillance in the region.