Theileria (T.) annulata stands out among Theileria species due to its severe impact on bovine health and productivity, highlighting the need for focused epidemiological research in Pakistan. To investigate the prevalence of T. annulata in district Pattoki, 188 blood samples were collected from cattle (n=131) and buffaloes (n=57) through convenient sampling method. All the samples were screened by microscopy for the presence of Theileria-like inclusion bodies and PCR for molecular confirmation of T. annulata, targeting the cytochrome b (cyt b) gene to estimate the prevalence of this pathogen. Moreover, the PCR products were sequenced and analyzed for possible variations in the cytochrome b gene using different bioinformatics tools. Microscopic examination revealed that out of 188 blood samples, 31 samples (16.49%) tested positive for Theileria while 50 samples were confirmed positive as T. annulata by PCR with an overall prevalence of 26.60%. The BLAST alignment revealed variations at multiple positions in the cytochrome b gene of T. annulata isolates. The sequence analysis showed 99.55% similarity between Cyt b/Pak 1 and Cyt b/Pak 4 while 100% similarity between Cyt b/Pak 2 and Cyt b/Pak 3. Cyt b/Pak 5 showed comparatively less similarity with other study sequences. A varying similarity of study isolates was found with the isolates of other neighboring countries including Japan (LC431535), China (KP731977), India (MG787979), Turkey (MK693128), Iran (MT812969), Egypt (PP920504), and Tunisia (KF732030). Among different risk factors, previous tick history, previous anti-theileria treatment, tick control and education status of the owner were found as significant (p<0.05) risk factors associated with the prevalence of theileriosis in bovines. It was concluded that T. annulata is a prevalent pathogen of bovines in the study area and is significantly related to various assumed risk factors. Moreover, variations in the sequences of the cytochrome b gene of T. annulata are evident, necessitating further study to explore any potential correlation with buparvaquone resistance in theileriosis.