Background: To address the clinical relevance of molecular detection of occult breast cancer in sentinel lymph nodes and nonsentinel axillary lymph nodes (ALN), we initiated the Minimally Invasive Molecular Staging of Breast Cancer (MIMS) trial, a multi-institutional prospective cohort study. This trial represents the first prospective cohort study in which a multimarker, real-time reverse transcription polymerase chain reaction (RT-PCR) analysis was applied to the detection of breast cancer micrometastases in ALN. Materials and Methods: Sentinel and/or nonsentinel ALN from 501 breast cancer subjects with T1-T3 primary tumors were analyzed by standard histopathology and multimarker, real-time RT-PCR analysis. Seven breast cancer-associated genes (mam, mamB, PIP, CK19, muc1, PSE, and CEA) known to be overexpressed in metastatic breast cancer compared with control lymph nodes were used. Follow-up data were collected for 5 years. Results: Of the 501 breast cancer subjects enrolled, 348 were node negative and completed the 5-year follow-up. Of these patients (n = 94), 27% demonstrated evidence of molecular overexpression. The 5-year relapse-free survival rate was 95.4% (95% confidence interval [95% CI], 92.4-97.2%). No single gene or combination of study genes was predictive of recurrence. Conclusions: The genes in this study panel failed to be predictive of clinical relapse. This may be a function of several factors: the low event rate at 5 years, the particular gene set, the methodology used for detection/analysis or that our original hypothesis was wrong and that the presence of positive marker signal by real-time RT-PCR is not associated with a worsened clinical outcome.