SUMMARY At the global population level, seasonal influenza A virus (IAV) evolution is characterized by repeated selective sweeps in which novel antigenic variants that escape antibody responses replace previously circulating strains. Despite this clear pattern at a population level, selection of antigenic variants is not typically seen within individuals with acute IAV infection. This apparent disconnect between evolutionary processes at population and within-host scales contributes to the challenge of predicting evolutionary outcomes. The processes that impede IAV selection within the host, and their potency, have not been examined systematically. To address this knowledge gap, we will test the central hypothesis that the genetic structure of within-host viral populations plays a major role in defining the likelihood that a beneficial mutation becomes fixed. In particular, we hypothesize that the fate of a highly fit variant virus is shaped by its initial prevalence in the inoculum (quantitative structure), the timing with which it arises de novo (temporal structure), its location within the infected tissue (spatial structure) and the presence or absence of competing variants (clonal interference). In turn, we predict that onward transmission of a beneficial variant will rely on a combination of these factors and the stringency of the transmission bottleneck. We furthermore propose that the severity of the bottleneck is modulated by pre-existing immunity in the host. To test these hypotheses, we will monitor the dynamics of mixed viral populations containing a seasonal IAV and one or two HA antigenic variants thereof within individual guinea pigs and between transmission partners. Partial immunity will be induced with vaccination to yield a model in which the antigenic variants have a selective advantage. Distinct sequence barcodes will be included in the HA and NA genes of each virus to allow sensitive detection of genetic bottlenecks. Tracking barcodes in both HA and NA will allow genetic linkage to be assessed. The wild type and antigenic variant viruses will be combined in vivo with various quantitative, temporal and spatial structures and then barcode dynamics will be monitored by sequencing. With this approach, we will perform a systematic evaluation of the consequences of population genetic structure for viral evolutionary dynamics. This well-controlled and systematic approach will allow identification of processes that impede selection within and between individual hosts, providing much needed insight into the forces shaping IAV evolution where it begins.