Airborne microorganisms in the upper troposphere and lower stratosphere remain elusive due to a lack of reliable sample collection systems. To address this problem we designed installed and flight-validated a novel Aircraft Bioaerosol Collector (ABC) for NASA s C-20A that can make collections for microbiological research investigations up to altitudes of 13.7 km. Herein we report results from the first set of science flights xe2 x80 x94four consecutive missions flown over the United States (US) from 30 October to 2 November 2017. To ascertain how the concentration of airborne bacteria changed across the tropopause we collected air during aircraft Ascent/Descent (0.3 to 11 km) as well as sustained Cruise altitudes in the lower stratosphere (~12 km). Bioaerosols were captured on DNA-treated gelatinous filters inside a cascade air sampler then analyzed with molecular and culture-based characterization. Several viable bacterial isolates were recovered from flight altitudes including Bacillus sp. Micrococcus sp. Arthrobacter sp. and Staphylococcus sp. from Cruise samples and Brachybacterium sp. from Ascent/Descent samples. Using 16S V4 sequencing methods for a culture-independent analysis of bacteria the average number of total OTUs was 305 for Cruise samples and 276 for Ascent/Descent samples. Some taxa were more abundant in the flight samples than the ground samples including OTUs from families Lachnospiraceae Ruminococcaceae and Erysipelotrichaceae as well as the following genera: Clostridium Mogibacterium Corynebacterium Bacteroides Prevotella Pseudomonas and Parabacteroides. Surprisingly our results revealed a homogeneous distribution of bacteria in the atmosphere up to 12 km. The observation could be due to atmospheric conditions producing similar background aerosols across the western US as suggested by modeled back trajectories and satellite measurements. However the influence of aircraft-associated bacterial contaminants could not be fully eliminated and that background signal was reported throughout our dataset. Considering the tremendous engineering challenge of collecting biomass at extreme altitudes where contamination from flight hardware remains an ever-present issue we note the utility of using the stratosphere as a proving ground for planned life detection missions across the solar system.