Alternaria species produce various sorts of toxic metabolites during their active growth and causes severe diseases in many plants by limiting their productivity. These toxic metabolites incorporate various mycotoxins comprising of dibenzo-α-pyrone and some tetramic acid derivatives. In this study, we have screened out total 48 isolates of Alternaria from different plants belonging to different locations in India, on the basis of their pathogenic nature. Pathogenicity testing of these 48 strains on susceptible tomato variety (CO-3) showed 27.08% of the strains were highly pathogenic, 35.41% moderately pathogenic and 37.5% were less pathogenic. Phylogenetic analysis showed the presence of at least eight evolutionary cluster of the pathogen. Toxins (TeA, AOH and AME) were isolated, purified on the basis of column chromatography and TLC, and further confirmed by the HPLC-UV chromatograms using standards. The final detection of toxins was done by the LC-MS/MS analysis by their mass/charge ratio. The present study develops an approach to classify the toxicogenic effect of each of the individual mycotoxins on tomato plant and focuses their differential susceptibility to develop disease symptoms. This study represents the report of the natural occurrence and distribution of Alternaria toxins in various plants from India.