Correct identification of fungi to species level is important because a specific epithet embodies a set of characters that enables us to predict, for example, the mycotoxin production of a species. Many small-spored Alternaria isolates have been misidentified due to inappropriate growth conditions and the use of spore size as the only identifying character. In this study 39 Alternaria isolates were grown under standardised conditions and subjected to chemical, morphological and physiological analyses. All isolates were extracted and analysed by HPLC–DAD. Analysis showed that both A. gaisen and A. longipes were able to produce altertoxin I, which has not previously been reported. The resulting metabolite profiles were subjected to cluster analysis and principal component analysis. A subset of the isolates was grown at five different temperatures. Colony colour and diameter were recorded and the diameter measurements were subjected to principal component analysis. Analysis of chemical and physiological data showed that the 39 isolates segregated into the same distinct groups that are morphologically identifiable as A. alternata, A. longipes or A. gaisen. The results showed that A. longipes, A. gaisen and A. alternata are different species that can be distinguished morphologically, physiologically and chemically. Therefore, the continued use of the name Alternaria alternata for A. longipes and A. gaisen is unwarranted and pathotypes should not be used.