An approach for determination of hepatitis C virus (HCV) quasispecies by end-point limiting-dilution real-time PCR (EPLD-PCR) is described. It involves isolation of individual coexisting sequence variants of the hypervariable region 1 (HVR1) of the HCV genome from serum specimens using a limiting-dilution protocol. EPLD-PCR applied to an HCV outbreak study provided insights into the epidemiological relationships between incident and chronic cases. When applied to samples from a longitudinal study of infected patients, HVR1 sequences from each sampling time-point were observed to group as distinct phylogenetic clusters. Melting peak analysis conducted on EPLD-PCR products generated from these patients could be used for evaluation of HVR1 sequence heterogeneity without recourse to clonal sequencing. Further, to better understand the mechanism of single-molecule PCR, experiments were conducted under optimal and suboptimal annealing temperatures. Under all temperature conditions tested, HVR1 variants from the major phylogenetic clusters of quasispecies could be amplified, revealing that successful HVR1 quasispecies analysis is not contingent to dilution of starting cDNA preparations to a single-molecule state. It was found that EPLD-PCR conducted at suboptimal annealing temperatures generated distributions of unique-sequence variants slightly different from the distribution obtained by PCR conducted at the optimal temperature. Hence, EPLD-PCR conditions can be manipulated to access different subpopulations of HCV HVR1 quasispecies, thus, improving the range of the quasispecies detection. Although EPLD-PCR conducted at different conditions detect slightly different quasispecies populations, as was shown in this study, the resulted samples of quasispecies are completely suitable for molecular epidemiological investigation in different clinical and epidemiological settings.