Chronic hepatitis C virus (HCV) infection exists as a complex mixture of genetically distinct viruses, commonly referred to as a “quasispecies.” Quasispecies complexity can vary substantially during the course of natural infection as a consequence of viral population “bottlenecking.” This occurs at the time of transmission from one individual to the next and during the course of chronic infection of an individual when adaptive immune responses eliminate certain viruses but allow others to escape and expand. Antiviral treatment with drugs that fail to eradicate virus can also lead to virus population bottlenecks and emergence of drug-resistant variants. Single genome sequencing (SGS) combined with mathematical modeling and phylogenetic inference is a recently described approach for characterizing the HCV quasispecies in unprecedented detail, allowing for the first time the retention of genetic linkage across genes and near full-length genomes and precise identification of transmitted/founder (T/F) genomes. Here, we describe the methodological approach to SGS and show how this strategy allows for the precise and unambiguous molecular identification of transmitted viruses as well as those that repopulate the body after drug or immune-mediated selective sweeps. This is an enabling experimental strategy that allows for a precise genetic, biologic, and antigenic characterization of HCV viruses that are responsible for transmission and persistence. Such an approach can be particularly valuable to future HCV vaccine design efforts, as it has been for human immunodeficiency virus type 1 (HIV-1).