Ticks are detrimental ectoparasites that consume the blood of humans and animals. They serve as crucial carriers for various parasitic diseases in mammals. Existing data indicates that the study of ticks infesting buffaloes in Babylon City is incomplete, and their presence and identification have not been fully examined. This work aimed to use microscopic and molecular approaches to isolate and identify tick species that infest buffaloes. The results revealed that 213 out of 1000 animals (21.3%) were found infested by ticks based on the clinical inspection and microscopic examination in different areas, including Mahaweli, Al-Qasim, Al Saddah, Kifl, Al-Musayyab, Al-Wardia, and Awfi, from September 2023 to January 2024. The microscopic findings found that buffaloes were only infested by Hyalomma spp. The infection rate in females was higher than males in the Al Saddah region. A molecular study using PCR and DNA sequencing techniques identified two genera of hard ticks, Rhipicephalus and Hyalomma, in all infested buffaloes. The sequencing technique demonstrated three species of the genus Hyalomma, including H. anatolicum, H. excavatum, H. scupense, while R. turanicus was only identified during this study. Interestingly, H. scupense was identified for the first time in Babylon province based on DNA sequencing. Overall, this is the first molecular study to identify tick species infesting buffaloes in Babylon province in Iraq, which recommends more hygienic measures to overcome the tick species of Rhipicephalus and Hyalomma.