Collagen I (Col1) fibers are a major structural component in the extracellular matrix of human breast cancers. In a preliminary pilot study, we explored the link between Col1 fiber density in primary human breast cancers and the occurrence of lymph node metastasis. Col1 fibers were detected by second harmonic generation (SHG) microscopy in primary human breast cancers from patients presenting with lymph node metastasis (LN+) versus those without lymph node metastasis (LN−). Col1 fiber density, which was quantified using our in-house SHG image analysis software, was significantly higher in the primary human breast cancers of LN+ (fiber volume=29.22%±4.72%, inter-fiber distance=2.25±0.45 μm) versus LN− (fiber volume=20.33%±5.56%, inter-fiber distance=2.88±1.07 μm) patients. Texture analysis by evaluating the co-occurrence matrix and the Fourier transform of the Col1 fibers proved to be significantly different for the parameters of co-relation and energy, as well as aspect ratio and eccentricity, for LN+ versus LN− cases. We also demonstrated that tissue fixation and paraffin embedding had negligible effect on SHG Col1 fiber detection and quantification. High Col1 fiber density in primary breast tumors is associated with breast cancer metastasis and may serve as an imaging biomarker of metastasis.