Tomato is an important vegetable crop infected by several viral diseases among them Tomato mosaic disease incited by Tomato mosaic virus (ToMV) is a major constraint for quality tomato production. Tomato growing majorly throughout the year in districts of Anantpur, Kurnool, and Chitoor in Southern Andhra Pradesh. The inoculum is readily available to make the disease a constant threat during all seasons. A survey conducted during the rabi season of 2017-2018 in major tomato growing areas observed that plants were infected with mosaic symptoms. The highest mean percent disease incidence (PDI) of mosaic symptoms on tomato was recorded in the Anantapur district (6.31%) followed by the Chittoor district (5.43%) and lowest in Kurnool district (4.62%). The higher PDI of tomato mosaic disease was reported in Kadiri mandal (6.83%) followed by Kalyanadurgam (6.5%) and lowest PDI at Pileru mandal (3.44%). Similarly, the highest PDI was reported in Hampapuram village (8.41%) followed by Ramiganivaripalle village (8.33%), and the lowest at Kalikiri village (299%). The rod-shaped viral particles were identified from infected samples on TEM assay. The 42.10% (40/95 samples) collected from 30 different major tomato-growing villages, were expressed HR responses on test plants (N. glutinosa). The DAC-ELISA assay with infected samples revealed that the mosaic eliciting agent was Tomato mosaic virus (ToMV) in South Andhra Pradesh.