Abstract Summary The exploration and comparison of genome organization is routinely used in the frame of genomic and phylogenomic analyses. As a consequence, in the past few years, various tools allowing visualizing genomic contexts have been developed. However, their use is often hampered by a lack of flexibility, particularly concerning associated databases input formats and figure customization. Here we present GeneSpy, a graphical user interface that allows the visualization and dynamic exploration of eukaryotic and prokaryotic annotated genomes. GeneSpy relies on user-friendly manageable local databases and allows the easy customization and production of figures in a multitude of formats. Availability and implementation GeneSpy is freely available at https://lbbe.univ-lyon1.fr/GeneSpy/ for Linux, Mac OS and Windows under CeCILL license (http://www.cecill.info/licences/). It is written in Python 2.7 and depends on Matplotlib, Tkinter and Sqlite libraries. Supplementary information Supplementary data are available at Bioinformatics online.