Hepatitis C virus (HCV) is a medically important RNA virus in the Flaviviridae family. It persists in chronically infected individuals by replicating in hepatocytes and by evolving as a genetically diverse "quasispecies" that evades host immune pressures. However, transmission, with its attendant population bottlenecking, represents a period of relative vulnerability and is of particular importance with respect to viral natural history, immunopathogenesis, treatment intervention, and vaccine development. A precise molecular characterization of HCV transmission and early diversification has not previously been possible. In this dissertation work, it was hypothesized that HCV genomes that are transmitted from one individual to the next giving rise to productive clinical infection (termed transmitted/founder or T/F genomes) could be unambiguously identified by single genome sequencing (SGS), mathematical modeling, and phylogenetic inference. This hypothesis was tested in cohorts of acutely infected human subjects with community acquired HCV infection and in human-to-human and human-to-chimpanzee HCV transmission pairs. The resulting data showed that HCV transmission was generally associated with a stringent population bottleneck and that early virus evolution was characterized by diversification of discrete, low diversity sequence lineages. These findings enabled an unambiguous phylogenetic inference of T/F genomes, a precise characterization of early molecular pathways of viral sequence evolution, and a refined estimate of the in vivo mutation rate of HCV, which was at least 5-fold lower than previously reported. These efforts further allowed for the molecular identification, cloning, and analysis of full-length T/F viral genomes, which like most HCV clones, were restricted in their in vitro replication capacity. Altogether, our findings provide a substantially enhanced molecular view of HCV transmission and early diversification in natural human infection and illustrate a novel experimental approach to the proteome-wide analysis of HCV that may aid future vaccine development efforts.