Free-living amoebae of the genus Acanthamoeba are the causative agents of severe sight-threatening infection of the cornea. This study was designed to characterize the genotype of 20 Acanthamoeba spp. isolates obtained from corneal scrapings of 183 suspected Acanthamoeba keratitis patients reporting to the Outpatient Department/Casualty Services of Dr R. P. Centre for Ophthalmic Sciences, All India Institute of Medical Sciences, New Delhi, India during the period 2011–2015. Corneal scrapings were inoculated onto 2 % non-nutrient agar plates overlaid with Escherichia coli and incubated at 30 °C for 15 days. Amongst 183 suspected Acanthamoeba keratitis patients, 29 were found culture-positive for Acanthamoeba spp. out of which 20 samples were established in axenic culture for molecular analysis. DNA was isolated and PCR assay was performed for the amplification of the diagnostic fragment 3 (DF3) (∼280 bp) region of the 18S rRNA gene from axenic culture of 20 Acanthamoeba spp. isolates. Rns genotyping was performed on the basis of variation in nucleotide sequences of the DF3 region of the 18S rRNA gene. In the phylogenetic analysis, 16 of the 20 isolates were found to be of prevalent genotype T4, two were of genotype T10 and the remaining two isolates were of unassigned genotypes. Hence, it was concluded that genotype T4 was found as the most predominant genotype involved in Acanthamoeba keratitis infections. Genotype T10, which had not been reported from India, was detected for the first time in two patients. Two isolates were found to be unique, which shared < 95 % homology with all the known genotypes (T1–T20) of Acanthamoeba spp.