Twister disease is a major disease of onion (Allium ascalonicum L.). The causal organisms of the anthracnose/twister disease of onion were characterized and identified using molecular markers to assess the diversity of the pathogens and the identity of the pathogen was confirmed by sequencing the amplified ITS rDNA region, and analysing in NCBI BLAST program and identified as C. gloeosporioides, C. acutatum, G. acutata , F. oxysporum and G. moniliformis. The phylogenetic tree showed that all five isolates of Colletotrichum spp.  Framed into 3 clusters. Since, there is no gene sequences of C. gloeosporioides  C. acutatum and F. oxysporum causing onion twister disease in the Genbank we have deposited our sequences Genbank. Specific DNA amplification of region CgInt was fairly consistent band of 450 bp for C. gloeosporioides and for C. acutatum it was 490 bp which was positive for all isolates of C. acutatum. For F. oxysporum amplification was at 550-570 bp. The products of the PCR ITS rDNA digestion with DraI revealed that no restriction sites were present. The digestion with HaeIII resulted in a characteristic pattern of three fragments in 19 C. gloeosporioides isolates. In 24 isolates of C. acutatum isolates and 25 isolates F. oxysporum showed four clusters.