Prox1 is a divergent homeodomain protein important for the development of the lens, retina, liver, pancreas, and lymphatic vasculature. Prox1 expression is highly upregulated in transformed hepatocytes and has been used as a marker to distinguish lymphatic from blood vasculature. We produced recombinant human Prox1 (amino acids 547-737) fused to glutathione S-transferase (GST) and used it to create two hybridomas, 5G10 and 4G10. Both of these hybridomas produced monoclonal antibodies able to detect Prox1 by immunofluorescence in lenses from diverse terrestrial vertebrates, including humans, rats, chickens, and lizards, although 5G10 was generally more sensitive in this application. Further, 4G10 was able to robustly detect endogenous and recombinant Prox1 in both cell and tissue extracts by Western blotting, while 5G10 was notably less sensitive for this purpose. These monoclonal antibodies will be useful for diverse studies on the role of Prox1 in both normal development and disease processes in terrestrial vertebrates.