Purpose The purpose of this study was to detect the lymphatic drainage pattern of internal mammary area and verify the concept of internal mammary sentinel lymph node (IM-SLN) in breast. Materials and Methods A small particle radiotracer ( 99m Tc-Dextran 40) was prepared and tested. 99m Tc-Dextran 40 was injected into intraparenchyma at the sound breast by a modified radiotracer injection technique. Subsequently, dynamic single-photon emission computed tomography (SPECT), computed tomography (CT), and SPECT/CT combination images were performed to identify the radioactive lymph vessels and internal mammary lymph nodes (IMLNs). The direction of lymph drainage and the location of the IMLNs were identified in the SPECT/CT imaging. Results The radiochemical purity of 99m Tc-Dextran 40 was > 95%. 99m Tc-Dextran 40 could drainage into first, second, and third lymph node and the radioactive lymph node could be detected by the γ detector in the animal experiment. After 99m Tc-Dextran 40 injecting into intraparenchyma, 50.0% cases (15/30) were identified the drainage lymphatic vessels and radioactive IMLNs by SPECT. The drainage lymphatic vessel was found from injection point to the first IMLN (IM-SLN) after 10.5±0.35 minutes radiotracer injection, and then 99m Tc-Dextran 40 was accumulated into the IM-SLN. The combination imaging of SPECT/CT showed the second IMLN received the lymph drainage from the IM-SLN. The lymphatic drainage was step by step in the internal mammary area. Conclusion The lymph was identified to drain from different regions of the breast to IM-SLN, and then outward from IM-SLN to other IMLN consecutively. It demonstrated the concept of the IM-SLN and provided more evidences for the application of internal mammary sentinel lymph node biopsy.