<p>Acoustic reporter genes (ARGs) have enabled imaging of gene expression with ultrasound, which provides high resolution access to deep, optically opaque living tissues. However, unlike their fluorescent counterparts, ARGs have so far been limited to a single ‘sound’, preventing multiplexed imaging of cellular states or populations. Here we use rational protein design and directed evolution to develop two new ARGs that can be distinguished from each other based on their acoustic pressure-response profiles, enabling ‘two-tone’ ultrasound imaging of gene expression. We demonstrate the utility of multiplexed ARGs for delineating bacterial cell species and cell states in vitro, and then apply them towards imaging distinct subpopulations of probiotics in the mouse gastrointestinal tract and of tumor-colonizing bacterial agents in vivo. Just as the first wavelength-shifted derivatives of fluorescent proteins opened a vivid world for optical microscopy, our next-generation acoustic proteins set the stage for a rich symphony of ultrasound signals from living subjects.</p>