We previously reported that D2-40 antibody identifies lymphatic endothelial cells (LECs) of lymphatic malformations (LM) with high specificity but only moderate sensitivity. The aim of this study was to compare the sensitivity and specificity of the markers Prospero-related homeobox gene-1 (Prox-1) and VEGFR3 to those of D2-40 for LECs in LM. Seventeen LM and 6 other vascular malformations with venous component (VM) were stained with D2-40, Prox-1, VEGFR3, CD31, and CD34 antibodies. The staining characteristics of vessels by each marker were assessed. Prox-1 and VEGFR3 specificity for LECs was examined by endothelial staining of VMs. Prox-1 and VEGFR3 stained substantially more large vessels than did D2-40 (15 of 17 cases). Small vessel staining was uniformly positive with all vascular markers except CD34, which did not stain lymphatic vessels. Eight cases had no or minimal D2-40 staining of large vessels, but the selected D2-40-vessels stained positive with VEGFR3, and 7 of 8 vessels were Prox-1 positive. Nine cases had variable D2-40 staining of large vessels; the selected D2-40 channels were all Prox-1 positive, and 8 of 9 were VEGFR3 positive. Two had rare vascular channels with no lymphatic marker stain but were positive for CD31 and CD34. Endothelial cells of VM had no Prox-1 but were VEGFR3/CD31/CD34 positive. VEGFR3 and Prox-1 antibodies have greater sensitivity for large lymphatic vessels than does D2-40. All lymphatic markers have high sensitivity for LECs of small lymphatic channels. Prox-1 has superior sensitivity and specificity to LECs. We recommend utilizing an immunohistochemical panel consisting of Prox-1, VEGFR3, CD31, and CD34 antibodies to differentiate lymphatic from venous malformations in pathologic practice.