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The objective of the present study was to detect the chosen nucleotide DNA or RNA sequences of the pathogens in ticks of domestic and wild animals of Kerala, South India based on molecular techniques. Among 602 ticks collected, 413 were from bovines (cattle and buffalo), 26 from goats, 101 from dogs and 62 from wild animals. Amblyomma integrum, Am. gervaisi, Dermacentor auratus, Haemaphysalis bispinosa, Ha. intermedia, Ha. shimoga, Ha. spinigera, Rhipicephalus annulatus, Rh. microplus, Rh. haemaphysaloides and Rh. sanguineus s.l. were identified from various domestic and wild animals of Kerala. The cDNA synthesized from the RNA isolated from fully or partially engorged adult female/nymphal ticks was used as template for the specific polymerase chain reactions (PCR). Out of 602 ticks examined, nucleotide sequences of pathogens were detected in 28 ticks (4.65%). The nucleotide sequences of tick-borne pathogens like Theileria orientalis, Babesia vogeli, Hepatozoon canis, Anaplasma marginale, An. bovis, Rickettsia sp. closely related to Ri. raoultii, Ri. massiliae, Ri. africae and Ri. slovaca were detected. The identification of the previously unreported nucleotide sequences of rickettsial pathogens from India is of particular interest due to their zoonotic significance. The phylogenetic analysis of the major piroplasm surface protein (MPSP) gene of T. orientalis amplified from Rh. annulatus ticks revealed that they were genetically close to type 7, which belong to the highly pathogenic Ikeda group.