Colletotrichum truncatum is an important exotic pathogen threat to the Australian lentil industry. Canadian isolates of C. truncatum from lentil were assessed for morphological and molecular differences against Australian and international isolates of C. truncatum from hosts other than lentil and other Colletotrichum species. Although similar in dimension, conidiospores from lentil C. truncatum were ellipsoidal and those from C. truncatum originating from Australian Glycine max, Xanthium occidentale and Arachis hypogaea were falcate in shape. Conidiospores of Australian C. trifolii, C. gloeosporioides and C. destructivum were cylindrical. Molecular analysis, using both RAPD markers and 18–25S rDNA sequences, demonstrated the genetic relatedness of C. truncatum isolates from the same host species and discriminated among isolates from lentil and other host species. The Canadian isolates of C. truncatum from lentil formed a single distinct cluster, separate from that of the C. truncatum isolates originating from other legume host species and other Colletotrichum species from within and outside Australia. Conserved nucleotide sequence was used to develop a sensitive and highly specific diagnostic marker for the Canadian lentil isolates. The marker detected the pathogen in as little as 0.1 ng of DNA from infected plant tissue and represents a major step towards preventing or delaying the introduction and establishment of lentil anthracnose in Australia.