The upper and lower respiratory tracts (URT and LRT) present distinct environments for influenza A virus (IAV) replication. Their differential features have major implications for viral evolutionary dynamics, transmission potential, and pathogenesis. To investigate the implications of differential viral replication in the URT and LRT, we assessed dispersal of IAVs throughout the guinea pig respiratory system. Guinea pigs were inoculated intranasally with a 300 μL volume to deliver inoculum to both the URT and LRT. Two strains were used to represent the circulating seasonal IAV lineages: influenza A/TX/50/2012 (H3N2) and influenza A/CA/07/2009 (H1N1). For the H1N1 virus, a genetically diverse barcode library enabled high-resolution tracking of viral dispersal. Infectious virus was consistently detected in the URT for both strains; however, only the H1N1 virus was detected in the LRT. To determine whether replication of the H1N1 virus in the LRT extends to other routes of infection, virus distribution was evaluated following infection via aerosol exposure or transmission. Infectious virus in lung homogenates was observed in both cases, confirming the LRT tropism of the H1N1 virus. Sequencing genetic barcodes revealed that diversity was largely maintained in nasal samples and trachea but was reduced upon dispersal to the lungs. This contraction of diversity was associated with increased distance and branching from the major airways, suggesting long-distance dispersal through the respiratory tract imposes within-host population bottlenecks. Together, these findings highlight how the distinct environments of the URT and LRT shape within-host IAV population dynamics.IMPORTANCEThe upper (URT) and lower (LRT) respiratory tracts create different conditions for influenza A virus (IAV) spread and evolution. We studied how the virus moves through guinea pigs´ airways after infection with H3N2 or H1N1 strains of IAV. Whether delivered intranasally, by aerosol or by transmission, the H1N1 virus replicated in the nasal cavity, trachea, and lungs. By contrast, the H3N2 virus stayed mostly in the nasal cavity. Genetic barcodes were used to track how the H1N1 virus moved and changed. The populations replicating in the nasal cavity and trachea maintained high diversity, but those sampled from the lungs showed low diversity. This bottlenecking effect was stronger for viral populations present deeper in the lungs. These findings show that the different environments of the URT and LRT strongly shape how influenza spreads and evolves inside a host.