Radiotherapy (RT) is an important component in the therapeutic approach to oncologic conditions. This study presents the investigative results on the impact of RT on lymphatic vessels and on the regenerative response of the lymphatic system in a mouse model. We first irradiated 3 groups of ten mice using brachytherapy in a single treatment of 20 Gy. We then performed morphological examination of the irradiated lymphatic vessels using an in vivo microscopic transillumination technique at 2, 4, and 6 weeks. Next we evaluated lymphatic flow using lymphoscintigraphy and in vivo microscopy at 6 to 11 weeks in: 10 additional mice following irradiation as above (IR), in 10 mice following incision of a lymphatic vessel (I), and in a non-treated control group of 10 mice (N). Intact lymphatic vessels were observed in all mice at 2, 4, and 8 weeks following the single dose of radiotherapy in the first group of mice and normal lymphatic flow was fully restored in the irradiated (IR) and incised (I) mice indicating that the reparative substitution lymphatic pathways are functioning normally. We found that following irradiation with one dose of 20 Gy, lymphatic vessels were not visibly damaged and also that lymphatic flow was consistently restored and substitutive lymphatic pathways formed.