Abstract Purpose: As a first-echelon nodal drainage site of breast cancer, the status of axillary lymph nodes (ALN) and internal mammary lymph nodes (IMLN) is valuable both for regional staging and treatment choice. The internal mammary sentinel lymph node biopsy (IM-SLNB) might provide minimally invasive staging and guide individual IMLN radiation and systemic therapy. In our earlier study, we have tried injecting radiotracer with a modified technique (periareolar intraparenchymal, high volume and ultrasound guidance), and got a high IMSLN visualization rate at 71.1%, which laid a technical feasibility for the further study and clinical application of IM-SLNB. A prospective multicenter study was designed to verify the repeatability of this high IMSLN visualization rate with the modified injection technique (CBCSG026, NCT03541278). High visualization rate and low false negative rate are prerequisites for the widespread of IM-SLNB. The question arises as to whether IMSLN detected with the modified technique should be considered as “true” IMSLN. The prospective, multicenter, clinical validation study of IM-SLNB followed by internal mammary lymph node dissection (IM-LND) was designed to verify the accuracy of IM-SLNB in patients with ALN positive breast cancer (CBCSG027, NCT03024463). Methods and Materials: CBCSG026 trial enrolled patients with both axillary negative and positive breast cancer. It was assumed that the visualization rate of IMSLN was 65% and the success rate of IM-SLNB was 90%. At the 5% significance level, a minimum of 350 patients were required to reach a power of 95% (two-sided test). CBCSG027 trial only enrolled axillary positive (cN+ with biopsy proving or cN0 with positive axillary SLN) breast cancer patients receiving mastectomy. IM-LND of the 1st to 3rd intercostal was performed immediately after IM-SLNB to verify its accuracy. At the 5% significance level and a power of 95% (two-sided test), at least 40 patients with positive IMLN were required (110~140 patients with the visualization of IM-SLN were scheduled for the enrollment.) Results: Seven centers in China enrolled more than three quarters patients before April 30, with 286/350 (81.7%) in CBCSG026 and 34/40 (85.0%) in CBCSG027. The overall IMSLN visualization rate is 68.2% (195/286) with a success rate of IM-SLNB of 94.9% (185/195). The overall IMLN positive rate is 42.0% (34/81) wih a false negative rate of 2.9% (1/34). Conclusion: Modified technique of radiotracer injection (periareolar intraparenchymal, high volume, and ultrasound guidance) could significantly improve the detection rate of IMSLN with very low false negative rate with the prospective, multicenter validation results, providing minimally invasive staging and guiding individual IMLN radiation and systemic therapy. Citation Format: Yongsheng Wang, Qing Lu, Shiguang Zhu, Wenhe Zhao, Guanglun Yang, Yuanxi Huang, Jiong Wu, Yanbin Liu, Xiao Sun, Pengfei Qiu. Prospective, multicenter, clinical validation study of internal mammary sentinel lymph node biopsy followed by internal mammary lymph node dissection with the modified injection technique (CBCSG026/27) [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P1-20-14.