Chagas disease remains the most important parasitic infection in Latin America. The aetiological agent, Trypanosoma cruzi (Kinetoplastida: Trypanosomatidae), is a complex vector-borne zoonosis transmitted in the faeces of hematophagous triatomine bugs (Hemiptera: Reduviidae: Triatominae), and maintained by mammalian reservoir hosts ranging from the southern United States to Argentinean Patagonia. In the absence of chemotherapy, infection is life-long and can lead to a spectrum of pathological sequelae ranging from subclinical to lethal cardiac and/or gastrointestinal complications in up to 30% of patients. T. cruzi displays remarkable genetic diversity, which has long been suspected to contribute to the considerable variation in clinical symptoms observed between endemic regions. Currently, isolates of T. cruzi can be assigned to a minimum of six stable genetic lineages or discrete typing units (DTUs) (TcI-TcVI), which are broadly associated with disparate ecologies, transmission cycles and geographical distributions. The principal mode of reproduction among T. cruzi strains is the subject of an intense, decades-old debate. Despite the existence of two recent natural hybrid lineages (TcV and TcVI), which resemble meiotic F1 progeny, a pervasive view is that recombination has been restrained at an evolutionary scale and is of little epidemiological relevance to contemporary parasite populations. The aim of this PhD project was to investigate T. cruzi genetic diversity through significant development of phylogenetic markers and their application to the characterization of natural parasite population structures and genetic exchange mechanisms. Multiple, single-copy, chromosomally-independent, nuclear housekeeping genes were assessed initially for their ability to allocate isolates to DTU-level, to facilitate higher resolution intra-lineage analyses and finally for their inclusion alongside additional targets in a standardized T. cruzi multilocus sequence typing (nMLST) scheme. For the immediate future, nuclear MLST, using a panel of four to seven nuclear loci, is a robust, reproducible and highly discriminatory method that has potential to become the new gold standard for T. cruzi DTU assignment. To investigate natural parasite population structures and uncover evidence of genetic exchange, a high resolution mitochondrial MLST (mtMLST) scheme, based on ten gene fragments, was developed and evaluated against current nuclear markers (multilocus microsatellite typing; MLMT) using isolates belonging to the oldest and most widely distributed lineage (TcI). Observations of gross nuclear-mitochondrial phylogenetic incongruence indicate that recombination is ongoing, geographically widespread and continues to influence natural populations, challenging the traditional paradigm of clonality in T. cruzi. Application of this combined nuclear-mitochondrial methodology to intensively sampled, minimally-subdivided TcI populations revealed extensive mitochondrial introgression within a disease focus in North-East Colombia as well as among arboreal transmission cycles in Bolivia. Failure to detect any reciprocal nuclear hybridization among recombinant strains ! 4 may be indicative of alternate, cryptic mating strategies in T. cruzi, which are challenging to reconcile with both in vitro parasexual mechanisms of genetic exchange described, and patterns of Mendelian allele inheritance among natural hybrid DTUs. High resolution genotyping of TcI populations was also undertaken to explore the interaction between parasite genetic heterogeneity and ecological biodiversity, exposing the significant impact human activity has had on T. cruzi evolution. Reduced genetic diversity, accelerated parasite dissemination between densely populated areas and mitochondrial gene flow between domestic and sylvatic populations, suggests humans may have played a crucial role in T. cruzi dispersal across the Bolivian highlands. Parallel reductions in genetic diversity were observed among isolates from the Brazilian Atlantic Forest, attributable to ongoing anthropogenic habitat fragmentation. By comparison domestic TcI isolates (TcIDOM) are divergent from their sylvatic counterparts, but also genetically homogeneous, and likely to have originated in North/Central America before distribution southwards. Molecular dating of Colombian TcIDOM clones confirmed that this clade emerged 23,000 ± 12,000 years, coinciding with the earliest human migration into South America. Lastly, Illumina amplicon deep sequencing markers were developed to explore the interaction between parasite multiclonality and clinical status of chronic Chagas disease. An unprecedented level of intra-host genetic diversity was detected, highlighting putative diversifying selection affecting antigenic surface proteases, which may facilitate survival in the mammalian host. In lieu of comparative genomics of representative T. cruzi field isolates, not yet a reality, as is the case with other more experimentally-tractable trypanosomatids, presented herein are some of the highest resolution genotyping techniques developed in T. cruzi to date, which have the potential to expand our current understanding of parasite genetic diversity and its relevance to clinical outcome of Chagas disease.