Podoplanin (PDPN)/T1alpha is expressed on lymphatic endothelial cells, type I alveolar cells of the lungs, and podocytes of the kidney. PDPN possesses three platelet aggregation-stimulating (PLAG) domains (PLAG1, PLAG2, and PLAG3) of the N-terminus and the PLAG-like domains (PLDs). We previously reported an anti-goat PDPN (gPDPN) monoclonal antibody (mAb), PMab-235, which was developed using the Cell-Based Immunization and Screening (CBIS) method. PMab-235 is very useful in flow cytometry, Western blotting, and immunohistochemical analyses; however, the binding epitope of PMab-235 remains to be elucidated. In this study, we investigated the epitopes of PMab-235 using enzyme-linked immunosorbent assay and immunohistochemistry. The results revealed that the critical epitope of PMab-235 produced by CBIS method is Arg75, Leu78, and Pro79 of gPDPN, which is included in PLD. The findings of our study can be applied to the production of more functional anti-gPDPN mAbs.