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 – four 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 sampler and then concentrated in the laboratory before molecular and culture-based characterization. Several culture-recovered bacterial isolates were captured 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 culture-independent analysis of bacteria and after removing the baseline signal (i.e., potential contaminants from controls) 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 the families Lachnospiraceae, Ruminococcaceae and Erysipelotrichaceae as well as the following genera: Clostridium, Mogibacterium, Corynebacterium, Bacteroides, Prevotella, Pseudomonas and Parabacteroides. Pseudomonas stutzeri was the only OTU identified at the species level that was significantly more abundant in flight samples than ground samples. Surprisingly, our results revealed homogenous distribution of bacteria in the atmosphere up to an altitude of 12 km. The finding could be due to atmospheric conditions producing similar background aerosols across the western U.S. as suggested by modeled back trajectories and satellite measurements. 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 mission across the solar system.