In routine surveillance and diagnostic testing, influenza virus samples are typically collected only from the upper respiratory tract (URT) due to the invasiveness of sample collection from the lower airways. Very little is known about virus variation in the lower respiratory tract (LRT) and it remains unclear if the virus populations at different sites of the human airways may develop to have divergent genetic signatures. We used deep sequencing of serially obtained matched nasopharyngeal swabs and endotracheal aspirates from four mechanically ventilated patients with influenza A/H3N2 infections. A physical barrier separating both compartments of the respiratory tract introduced as part of the medical procedures enabled us to track and compare the genetic composition of the virus populations during isolated evolution in the same host. Amino acid variants reaching majority proportions emerged during the course of infection in both nasopharyngeal swabs and endotracheal aspirates, and amino acid variation was observed in all influenza virus proteins. Genetic variation of the virus populations differed between the URT and LRT and variants were frequently uniquely present in either URT or LRT virus populations of a patient. These observations indicate that virus populations in spatially distinct parts of the human airways may follow different evolutionary trajectories. Selectively sampling from the URT may therefore fail to detect potentially important emerging variants. ImportanceInfluenza viruses are rapidly mutating pathogens that easily adapt to changing environments.Although advances in sequencing technology make it possible to identify virus variants at very low proportions of the within-host virus population, several aspects of intrahost viral evolution have not been studied because sequentially collected samples and samples from the lower respiratory tract are not routinely obtained for influenza surveillance or clinical diagnostic purposes. Importantly, how virus populations evolve in different parts of the human respiratory tract remains unknown. Here we used serial clinical specimens collected from mechanically ventilated influenza patients to compare how virus populations develop in the upper and lower respiratory tract. We show that virus populations in the upper and lower respiratory tract may evolve along distinct evolutionary pathways, and that current sampling and surveillance regimens likely capture only part of the complete intrahost virus variation.