In this study, 14 isolates of Acanthamoeba from both clinical and environmental sources belonging to seven different species were assayed for tolerance of high osmotic pressure, temperature tolerance, extracellular proteases, and cytopathic effects (CPE) on immortalized rabbit corneal epithelial cells. On the basis of the results, amoeba isolates were divided into pathogenic and nonpathogenic groups. Ribosomal DNA sequencing was performed on these isolates. Phylogenetic relationships revealed that all the pathogenic strains tested clustered together as one group, while nonpathogenic strains clustered into other groups. Sequence comparisons with previously published sequences determined that among the six new pathogenic isolates used in this study, five belong to T4 genotype and one to T11. This is the first report of a T11 genotype being found in Acanthamoeba keratitis.