The emergence of nanopore-based sequencers greatly expands the reach of sequencing into low-resource field environments, enabling in situ molecular analysis. In this work, we evaluated the performance of the MinION DNA sequencer (Oxford Nanopore Technologies) in-flight on the International Space Station (ISS), and benchmarked its performance off-Earth against the MinION, Illumina MiSeq, and PacBio RS II sequencing platforms in terrestrial laboratories. The samples contained mixtures of genomic DNA extracted from lambda bacteriophage, Escherichia coli (strain K12) and Mus musculus (BALB/c). The in-flight sequencing experiments generated more than 80,000 total reads on with mean 2D accuracies of 85 to 90%, mean 1D accuracies of 75 to 80%, and median read lengths of approximately 6,000 bases. We were able to make directed assemblies of the ~4.7 Mb E. coli genome, ~48.5 kb lambda genome, and a representative M. musculus sequence (the ~16.3 kb mitochondrial genome), at 100%, 100%, and 96.7% pairwise identity, and de novo assemblies of the lambda and E. coli genomes solely with yielded 100% and 99.8% genome coverage, respectively, at 100% and 98.5% pairwise identity. Across all surveyed metrics (base quality, throughput, stays/base, skips/base), no observable decrease in MinION performance was observed while sequencing DNA in space. Simulated runs of in-flight nanopore data using an automated bioinformatic pipeline demonstrated the feasibility of real-time sequencing analysis and metagenomic identification of microbes in space. Additionally, cloud and laptop based-assembly illustrated the plausibility of automated, de novo genomic assembly from nanopore data on the ISS. Applications of sequencing for space exploration include infectious disease diagnosis, environmental monitoring, evaluating biological responses to spaceflight, and even potentially the detection of extraterrestrial life on other planetary bodies.