OBJECTIVE Acute Hemorrhagic conjunctivitis (AHC) is associated with CV A24v. Recently there was a severe outbreak of conjunctivitis in months of July and August, 2023 in India. This study emphasizes the identification of the distinct mutations in the CV A24v strains, which were isolated during the AHC outbreak and could have potentially played a role in the high transmission of AHC in India during the 2023 outbreak. METHODS A total of 71 conjunctivitis patients aged 1-75 years comprising 47 males and 24 females who attended Ophthalmology department of a tertiary care hospital of easternIndia were studied.RNA was extracted from all conjunctival swab samples and converted into cDNA. Subsequently, the viral 5' UTR was amplified and the PCR positive samples were subjected to sequencing. The newly isolated viral 5' UTR sequences were aligned with other worldwide sequences using the Clustal W tool to conduct mutational analysis. A phylogenetic tree was built using the MEGA software for viral genotype identification. RESULTS All of the current outbreak strains belonged to genotype IV of CV A24v. The present outbreak strains formed a distinct clade in the phylogenetic tree and were different from previously reported Indian strains. Two persistent mutations, specifically in domain IV (T213C) and domain V (C475T), were exclusively detected within the internal ribosome entry site (IRES) of the 5' UTR of the current strains causing the outbreak. These two alterations have previously been shown to impact the virulence of another enterovirus (CV B3), but they have not been described in CV A24v until now. CONCLUSIONS Finding of the present study highlights the possibility and the significance of the aforementioned two mutations in enhancing the transmissibility of the newer CV A24v strains. Hence, these two distinct mutations should be investigated further for developing antiviral therapies to combat future AHC outbreaks associated with CVA 24v.