Study was undertaken in a theileriosis-endemic region of India during May 2018 to April 2019 among milch cows. Blood samples collected from apparently healthy (n = 65) and Theileria-suspect cows (n = 65) were screened against T. annulata and T. orientalis infection by SYBR Green‒based real time PCR using primers designed from the isolates of study area. Cows having single infection with T. annulata with/without clinical signs of inappetence, low milk yield, pale mucous membranes, fever, enlarged prescapular lymph node, soil licking, panting, coughing, salivation and lachrymation were subjected to further investigation where parasitaemia and piroplasms per 1000 erythrocytes ranged from 1.6 × 10 7 to 1.2 × 10 8 parasites/mL of blood and 3-24 piroplasms in moderate group (16/65), 4.4 × 10 8 to 6.9 × 10 9 parasites/mL of blood and >88 piroplasms in severe group (30/65) and 1.6 × 10 4 to 5.5 × 10 6 parasites/mL of blood and 0-1 piroplasms in asymptomatic or carriers (17/65), respectively. Study unfolded significant difference in T. annulata parasitaemia among apparently healthy and ill cows. Phylogenetic analysis of our T. annulata isolates (NCBI accession numbers MN098316, MN098317 and MN098318) exhibited maximum similarity with the isolates detected in other parts of India.