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Acanthamoeba is a pathogenic protist that causes a sight-threatening eye infection, Acanthamoeba keratitis (AK). The central focus of this research is to analyze the genetic relationships between Acanthamoeba isolates and how that contributes to pathogenesis. Acanthamoeba is a diverse genus with more than 18 genotypes based on the 18S ribosomal RNA gene. DNA barcoding has suggested that the mitochondrial cytochrome oxidase c subunit 1 (COI) gene can be accurately used to identify a eukaryotic organism. As part of understanding more about the phylogenetic structure of the genus of Acanthamoeba, representative isolates of most of the genotypes were analyzed using their COI genes. The phylogenetic relationships based on the COI gene were nearly identical to those produced using the 18S rRNA gene. This allows COI to be used as a reasonable substitute to the 18S rRNA gene. This project found an alternative method of classifying Acanthamoeba and allowed Acanthamoeba phylogenetics to be examined at more than one locus. A majority of this research focused on the ongoing Acanthamoeba keratitis outbreak in Chicago, Illinois since 2002. The dramatic increase of AK cases has occurred following EPA mandated water treatment changes which have been hypothesized to have increased microorganisms in the water system. To understand the phylogenetic structure of Acanthamoeba within this outbreak, a multilocus sequence typing (MLST) protocol was created. This project involved identifying and developing sequencing methods for five highly conserved housekeeping genes in Acanthamoeba. Sequences from these genes were used to determine the degree of variability amongst Acanthamoeba as well as to further our understanding of sub-lineages within the most common genotype, T4. In addition to the Chicago AK review, three surveys were conducted to elucidate the diversity of environmental Acanthamoeba from Chicago, Sonora, Mexico and on an Ohio farm. These surveys combined with the MLST study demonstrate how diverse the genus of Acanthamoeba is, regardless of source. Acanthamoeba are also known to contain potentially pathogenic endosymbionts including Legionella and Pseudomonas. The presence of endosymbionts has been shown to increase the pathogenicity of Acanthamoeba. To determine if endosymbionts in Acanthamoeba could contribute to the Chicago outbreak, Acanthamoeba was analyzed for the presence of Legionella and Pseudomonas. More than 50% of Acanthamoeba from Chicago contained one or both bacterial genera and these bacteria were located intracellularly in Acanthamoeba. We hypothesize that increases in microorganisms in the Chicago water systems increased the opportunity for Acanthamoeba to associate with pathogenic bacteria. Future research will examine the mechanism that these bacteria use to increase Acanthamoeba’s virulence as well as monitoring if diseases associated with these bacteria are also increasing. Together, these projects are meant to further our understanding on the diversity of the genus Acanthamoeba and the underlying factors of Acanthamoeba’s virulence.