The present study was designed to report the prevalence of spotted fever group Rickettsia and Anaplasma in ticks from Pakistan. To address this knowledge gap, ticks were collected from October 2019 to November 2020 from livestock hosts. 390 ticks from Punjab, Khyber Pakhtunkhwa, and Islamabad were investigated for the presence of Rickettsia and Anaplasma. The collected ticks were subjected to molecular studies for detection and characterization of spotted fever group Rickettsia and Anaplasma in ticks from Pakistan. PCR amplification of the ompA gene was used for detection of Rickettsia and portions of the 16S rDNA gene for detection of Anaplasma. Nine species of ticks were tested. 7/390 (2.58%) of ticks were positive for Rickettsia. Rickettsia spp. were detected in Haemaphysalis punctata, Hyalomma anatolicum, Hyalomma scupense, Rhipicephalus microplus, and Rhipicephalus sanguineus. Unknown Rickettsia was detected in Hy. scupense. 57 (14.6%) ticks were also positive for Anaplasma spp. Anaplasma ovis was detected in Hy. anatolicum, Hy. scupense, Hy. excavatum, Rhipicephalus decoloratus, R. microplus, and R. sanguineus. Anaplasma marginale was detected in Hy. anatolicum, Hy. scupense, R. microplus, R. decoloratus, and R. sanguineus. The Anaplasma sequences obtained from this experiment were 99–100% similar to those of documented strains. This study provides information and confirms the presence of spotted fever group Rickettsia and Anaplasma spp. in different tick species. It also highlights the need for control programs to prevent health risks. Further investigation to determine the prevalence and disease burden of these pathogens in Pakistan is necessary.