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Red rot disease is the most destructive disease of sugarcane in Bangladesh and causes major financial losses to the sugarcane growers. This study was carried out to characterize the Colletotrichum falcatum isolates of sugarcane collected from different regions in Bangladesh based on morphological, molecular characteristics. The four genes namely internal transcribed spacer (ITS-rDNA), beta-tubulin, Actin, and glaceraldehydes -3 phosphate dehydrogenase (GAPDH) sequences showed a 99-100% similarity to the conserved gene sequences in the GenBank database. The phylogenetic analysis of the four genes revealed that C.falcatum isolates from Bangladesh were clustered in the same clade and had no geographical distinctions differed from other countries' isolates. ISSR markers determined the low genetic diversity of the C. falcatum isolates in Bangladesh. The virulence levels of the 41 C. falcatum isolates corresponding to disease reaction on the host plant were categorized into three main groups viz. virulent, moderately virulent, and least virulent. The seven sugarcane differential hosts were classified as resistant, moderately resistant, and susceptible.