Background: The exact lymphatic drainage pattern of the breast hasn't been explained clearly. The aim of this study was to investigate the sentinel lymphatic channels (SLCs) in the cancerous breast. Whether the type of SLCs influenced the detection rate and false-negative rate of SLNB was also assessed. Methodology and Principal Findings: Mimic SLNB was performed in 110 early-stage breast cancer patients with subareolar injection of blue methylene dye intraoperatively. Postoperatively, 110 specimens of modified radical mastectomy were examined for all blue SLCs after additional injection of methylene dye in peritumoral parenchyma. Interestingly, three types of SLCs, including superficial sentinel lymphatic channel (SSLC), deep sentinel lymphatic channel (DSLC), and penetrating sentinel lymphatic channel (PSLC) were found in 107 patients. Six lymphatic drainage patterns based on the three types of SLCs were observed in these 107 patients. The proportions of the drainage pattern SSLC, DSLC, PSLC, SSLC+DSLC, SSLC+PSLC, and DSLC+PSLC in the breast were 43%, 0.9%, 15.9%, 33.6%, 3.7% and 2.8%, respectively. The lymphatic drainage pattern in the breast was a significant risk factor for unsuccessful identification of sentinel lymph nodes (P<0.001) and false-negatives in SLNB (P = 0.034) with the subareolar injection technique. Conclusions: Three kinds of SLCs are the basis of six lymphatic drainage patterns from the breast to the axilla. The type of SLCs is the factor influencing the detection rate and false-negative rate of SLNB. These findings suggest the optimal injection technique of the combination of superficial and deep injection in SLNB procedures. Future clinical studies are needed to confirm our novel findings. © 2012 Wang et al.