The mechanisms of lymphangiogenesis have been increasingly understood in recent years. Yet, the contribution of lymphangiogenesis versus lymphatic cooption in human tumors and the functionality of tumor lymphatics are still controversial. Furthermore, despite the identification of lymphatic endothelial cell (LEC) markers such as Prox1, podoplanin, LYVE-1, and VEGFR-3, no activation marker for tumor-associated LECs has been identified. Applying double-staining techniques with established LEC markers, we have screened endothelial cell differentiation antigens for their expression in LECs. These experiments identified the sialomucin CD34 as being exclusively expressed by LECs in human tumors but not in corresponding normal tissues. CD34 is expressed by LYVE-1(+)/podoplanin(+)/Prox1(+) tumor-associated LECs in colon, breast, lung, and skin tumors. More than 60% of analyzed tumors contained detectable intratumoral lymphatics. Of these, more than 80% showed complete co-localization of CD34 with LEC markers. In contrast, LECs in all analyzed normal organs did not express CD34. Corresponding analyses of experimental tumors revealed that mouse tumor-associated LECs do not express CD34. Taken together, these experiments identify CD34 as the first differentially expressed LEC antigen that is selectively expressed by tumor-associated LECs. The data warrant further exploration of CD34 in tumor-associated LECs as a prognostic tumor marker.