MOTIVATION: Viruses rapidly evolve due to their error-prone genome replication, and identifying which mutations are selected for during evolution is critical for virus surveillance efforts. Here we introduce a scatter plot tool (AAScatterPlot) that easily shows the selection and avoidance of certain protein mutations based on biochemical properties. We demonstrate its utility for monitoring the evolution of H9 avian influenza viruses from China between 2005 and 2015, particularly at the hemagglutinin (HA) proteolytic cleavage site (PCS) that can affect virus activation and pathogenicity. RESULTS: Given genome sequences, the AAScatterPlot tool compacts into a single plot, information about the hydropathy index, Van der Waals volume, chemical property and occurrence frequency of amino acid residues. The tool also shows the range of residues that could arise from a single point mutation in the genome, which can then be compared against the observed residues to identify mutation constraints. Through this approach, we found that the 2nd position towards the N-terminus side of the HA PCS (P2 position) avoided hydrophobic residues, whereas the P3 position avoided hydrophilic residues. AVAILABILITY AND IMPLEMENTATION: AAScatterPlot is available at https://github.com/WhittakerLab/AAScatterPlot. CONTACT: [email protected]. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.