Abstract: Ten isolates of A. alternata causing leaf blight disease of onion were collected from ten different conventional onion growing areas of Tamil Nadu and their pathogenicity was established. The species was identified by ICAR as Alternaria alternata (ITCC-Indian Type Culture Collection No. 5470). All the isolates produced light brown conidia with muriform shape, but the isolates varied in the size (length, width and beak length) of the conidia, the number of cells per conidium and sporulation times. Among the solid media tested host leaf extract agar and modified czapek's dox medium increased the growth of mycelium followed by potato dextrose agar medium and carrot agar medium. The maximum growth was observed in maltose followed by glucose, sucrose and fructose. Among the nitrogen sources, potassium nitrate followed by sodium nitrate and ammonium molybdate increased the growth of mycelium. Low pH (4-4.5) was found as ideal for the growth of A. alternata, while the minimum growth was observed in pH 9.0, irrespective of isolates. The conidia of A. alternata isolates germinated at 46°C.