Abstract Motivation An abundance of new reference genomes is becoming available through large-scale sequencing efforts. While the reference FASTA for each genome is available, there is currently no automated mechanism to query a specific sequence across all new reference genomes. Results We developed ACES (Analysis of Conservation with an Extensive list of Species) as a computational workflow to query specific sequences of interest (e.g. enhancers, promoters, exons) against reference genomes with an available reference FASTA. This automated workflow generates BLAST hits against each of the reference genomes, a multiple sequence alignment file, a graphical fragment assembly file and a phylogenetic tree file. These data files can then be used by the researcher in several ways to provide key insights into conservation of the query sequence. Availability and implementation ACES is available at https://github.com/TNTurnerLab/ACES Supplementary information Supplementary data are available at Bioinformatics online.