A new banana leaf spot disease (named as exserohilum leaf spot) caused by Exserohilum rostratum was found in Guangxi, China during a series of surveys on fungal species causing banana leaf diseases between 2005–2009. Three single spore derived isolates (CLER09, D087 and JL05) were obtained from diseased banana leaves and the pathogenicity of each isolate to banana plants was confirmed by inoculation tests based on Koch’s postulates. Three distinctive types of conidia (A, B and C) were produced by all isolates when subjected to a range of light environments. The isolates were identified as members of E. rostratum based on their morphological characters as well as rDNA-ITS (internal transcribed spacer) sequences. The effects of temperature and pH on vegetative growth and sporulation of the E. rostratum isolates were also characterized during this study. A similar host spectrum among the isolates was also observed in the host range tests on 128 plant species covering 47 families using a detached leaf inoculation technique.