The lymphatic system plays a key role in tissue homeostasis, fatty acid transport, and immune surveillance. Pathologically, dysfunction of the lymphatic system results in edema, and increased lymphangiogenesis can contribute to tumor metastasis. Lymphatic vessels are composed of lymphatic endothelial cells (LECs) that can be identified by distinct marker molecules such as Prox-1, podoplanin, VEGFR-3 and LYVE-1. Primary LECs represent a valuable tool for the study of basic functions of the lymphatic system. However, their isolation remains a challenge, particularly if rodent tissues are used as a source. We developed a method for the isolation of rat dermal LECs from the skin of newborn rats based on sequential enzymatic digestion with trypsin and Liberase followed by flow cytometric sorting using LYVE-1 specific antibodies. Cells isolated according to this protocol expressed the lymphatic markers Prox-1, podoplanin, LYVE-1 and VEGFR-3, and displayed an endothelial-like morphology when taken into culture. These primary cells can be used for studying lymphatic biology in rat models, and the protocol we describe here therefore represents an important extension of the experimental repertoire available for rats and for modeling the human lymphatic system.