The spread of tumor cells via lymphatic vessels to the lymph nodes is an important indicator of malignancy. However, previous markers used to identify lymphatic endothelium gave ambiguous results in immunohistochemical analyses with paraffin-embedded tissues. In this study, we attempted to prepare a polyclonal antibody against human lymphatic vessel endothelial hyaluronan receptor-1 (LYVE-1) for detecting lymphatic vessels using immunohistochemistry. The antibody was raised against a region near the transmembrane anchor of LYVE-1 in New Zealand white rabbits. Immunostainings with anti-LYVE-1 and von Willebrand factor antibodies were performed in various normal and pathological tissues. LYVE-1 expression was confined to the endothelial surface of lymphatic vessels but was not found in the endothelium of blood vessels, which were positive for von Willebrand factor. Our LYVE-1 polyclonal antibody was useful for the identification of small lymphatic vessels in normal human tissues. In addition, the immunostaining enabled us to distinguish lymphatic invasion by malignant tumor cells from blood vessel invasion using paraffin-embedded sections. In conclusion, our polyclonal antibody against the transmembrane anchor of the peptide can be used to detect human lymphatic vessels under various conditions.