Structural birth defects are the leading cause of infant mortality in the US and Europe. Among these, congenital heart disease (CHD) is the most common. Historically, Xenopus, mouse, and pig have provided models for CHD. However, it remains unknown what proteins and pathways are conserved between these species and human. Furthermore, the proteome driving the differences between three-chambered (Xenopus) and four-chambered (mammalian) hearts is unknown. Comparative proteomics of heart tissue from species at different evolutionary points can reveal molecular processes underlying heart function. We examined heart tissue proteomes of Xenopus tropicalis, Xenopus laevis, Mus musculus, and Sus scrofa and assessed protein expression changes in the context of pathways and protein complexes, and enrichment of corresponding genes in human heart diseases.