Phenotypic and genetic polymorphism was studied amongst four Theileria annulata isolates collected from three different parts of India. Amongst various markers studied for the comparison of growth characteristics of schizont cell lines established from these isolates, viability, non-viability counts and nitric oxide (NO) production showed significant variation. A negative correlation was observed between NO production and mRNA expression for TNF-alpha, a potent proinflammatory cytokine related to the pathogenesis of the disease. Phenotypic polymorphism was also revealed by T. annulata schizont-specific monoclonal antibodies (Mabs), viz. 1C7, 1E11, 2G2 and EU-106, which recognized variable number of cells in indirect fluorescent antibody and indirect immunoperoxidase tests, when tested against the four T. annulata isolates collected from India. Genetic polymorphism was recognized amongst the four isolates by restriction digestion analysis of Tams-1 gene PCR products. These observations revealed that the four isolates of T. annulata are different from each other and might be expressing different antigenic determinants on their cell surface.