AbstractMotivationHuge datasets containing whole-genome sequences of bacterial strains are now commonplace and represent a rich and important resource for modern genomic epidemiology and metagenomics. In order to efficiently make use of these datasets, efficient indexing data structures—that are both scalable and provide rapid query throughput—are paramount.ResultsHere, we present Themisto, a scalable coloredk-mer index designed for large collections of microbial reference genomes, that works for both short and long read data. Themisto indexes 179 thousandSalmonella entericagenomes in 9 h. The resulting index takes 142 gigabytes. In comparison, the best competing tools Metagraph and Bifrost were only able to index 11 000 genomes in the same time. In pseudoalignment, these other tools were either an order of magnitude slower than Themisto, or used an order of magnitude more memory. Themisto also offers superior pseudoalignment quality, achieving a higher recall than previous methods on Nanopore read sets.Availability and implementationThemisto is available and documented as a C++ package athttps://github.com/algbio/themistoavailable under the GPLv2 license.