Anthracnose, caused by Colletotrichum sp., is a serious problem of lupins (Lupinus spp.) worldwide. Morphological characters and molecular markers were used to characterize 43 Colletotrichum isolates from lupins, 8 isolates from other hosts, and 18 reference isolates representing related Colletotrichum spp., to assess the pathogen diversity and resolve its taxonomy. All lupin Colletotrichum isolates tested positive with C. acutatum-specific polymerase chain reaction (PCR) and did not test positive with C. gloeosporioides-specific PCR. Spore shape and colony diameter as well as insensitivity to benomyl grouped the lupin anthracnose isolates closer to C. acutatum than to C. gloeosporioides. Analysis of internal transcribed spacer (ITS) sequences of 57 Colletotrichum isolates grouped all lupin isolates with C. acutatum and distinct from C. gloeosporioides. Further, tub2 and his4 sequences revealed groups concordant with ITS, reducing the excessive dependence on the latter. Arbitrarily primed-PCR and amplified fragment length polymorphism analyses revealed intraspecific subgroups, but neither was useful to decipher species level relationships. ITS, tub2, and his4 results strongly support designating lupin anthracnose pathogen as C. acutatum or its subspecies. Most Colletotrichum isolates from lupins from worldwide locations are genetically homogeneous and form a distinct subgroup within C. acutatum. Present results also underline the potential of the C. acutatum-specific PCR for routine pathogen diagnosis.