An adult Indian buffalo (Bubalus bubalis) presented with corneal opacity, irritation, and excessive lacrimation from the left eye in the Referral Veterinary Polyclinic-Teaching Veterinary Clinical Complex (RVC-TVCC), Indian Veterinary Research Institute, Izatnagar. Clinical examination revealed a whitish thread-like worm in the left eye’s anterior chamber. The worm was surgically removed from the eye with supportive nerve blocks. Light microscopy was used for parasite morphological identification, which provided insight into the worm as female Setaria sp. Genomic DNA was isolated, and polymerase chain reaction amplification of 12S rRNA was conducted for molecular confirmation of the parasite. The amplicon was sequenced and analysed by bioinformatics software. Sequence data showed an amplicon size of 243 bp. Phylogenetic analysis with reference data from the NCBI Genbank database revealed the worm was S. digitata, with a similarity of 99.17%. The common predilection site of S. digitata is in the peritoneal cavity of natural hosts like cattle and buffalo and is mostly non-pathogenic. The aberrant migration of the parasite larva to the brain and eye commonly occurs in goats, sheep, and horses, causing clinical conditions like cerebrospinal nematodiasis (lumbar paralysis) and ocular setariasis, respectively. Nevertheless, until now, there have been no reports of ocular setariasis in buffalo. This report is the first unusual occurrence of ocular setariasis in buffalo and its molecular confirmation and phylogenetic analysis using 12S rRNA.