Changing environmental conditions, including those caused by human activities, reshape biological communities through both loss of native species and establishment of non-native species in the altered habitats. Dynamic interactions with the abiotic environment impact both immigration and initial establishment of non-native species into these altered habitats. The repeated emergence of disease systems in urban areas worldwide highlights the importance of understanding how dynamic migratory processes affect the current and future distribution and abundance of pathogens in urban environments. In this study, we examine the pattern of invasion of Trypanosoma cruzi—the causative agent of human Chagas disease—in the city of Arequipa, Peru. Phylogenetic analyses of 136 T. cruzi isolates from Arequipa and other South American locations suggest that only one T. cruzi lineage established a population in Arequipa as all T. cruzi isolated from vectors in Arequipa form a recent monophyletic group within the broader South American phylogeny. We discuss several hypotheses that may explain the limited number of established T. cruzi lineages despite multiple introductions of the parasite.