The beta scanner and pattern matching software integrated in the automated microbiology identification system (AMBIS) were used to assess the relatedness of isolates of Colletotrichum acutatum, C. kahawae, C. fragariae, C. gloeosporioides and C. musae based on mitochondrial DNA restriction fragment length polymorphisms. The dendrograms generated reflected the intra-specific variation and inter-specific relationships of these species as determined by other previously reported methods. The adaptability of AMBIS as a rapid method for assessing genetic relatedness of fungal species based on DNA polymorphisms is discussed.