2A peptides are sequences that trigger co-translational peptide-bond skipping. This unusual and efficient activity allows for the production of multiple proteins from a single open reading frame. Accordingly, 2A peptides have become commonly used throughout biomedical research. Despite their widespread use, little is known about the prevalence of these sequences throughout viruses and eukaryotes. Bioinformatics analysis greatly expanded the predicted number of 2A peptides known by over an order of magnitude. These sequences were shown to fall into two classes: the previously known Class A and a novel Class B. Testing the peptide-bond skipping activity of select sequences from both classes revealed the majority are functional in HEK293T cells. Further testing and analysis of the novel Class B showed that both the position and identity of amino acids within the sequence are critical for peptide-bond skipping. These results not only reveal a new class of 2A peptides but demonstrate that 2A peptides are more widespread in both viruses and eukaryotes than previously thought. The form and content of the abstract are approved. I recommend its publication.