Background: The standard of care for stage 1 NSCLC is upfront surgery followed by surveillance. However, 20-30% of stage 1 NSCLC recur. There is an unmet need to identify individuals likely to recur who would benefit from frequent monitoring and aggressive cancer treatments. Collagen 1 (Col1) fibers detected by second harmonic generation (SHG) microscopy are a major structural component of the extracellular matrix (ECM) of tumors that play a role in cancer progression. Method: We characterized Col1 fibers with SHG microscopy imaging of surgically resected stage 1 NSCLC. Gene expression from RNA sequencing data was used to validate the SHG microscopy findings. Results: We identified a significant (p ≤ 0.05) increase in the Col1 fiber volume in stage 1 NSCLC that recurred. The increase in Col1 fiber volume was supported by significant increases in the gene expression of Col1 in invasive, compared to noninvasive, lung adenocarcinoma. Significant differences were identified in the gene expression of other ECM proteins, as well as CAFs, immune checkpoint markers, immune cytokines, and T-cell markers. Conclusion: Col1 fiber analysis can provide a companion diagnostic test to evaluate the likelihood of tumor recurrence following stage 1 NSCLC. The studies expand our understanding of the role of the ECM in NSCLC recurrence.