Accurate stratification of tumors is imperative for adequate cancer management. In addition to staging, morphologic subtyping allows stratification of patients into additional prognostic groups. In this study, we used an image-based computational method on pan-cytokeratin IHC stainings to quantify tumor fragmentation (TF), a measure of tumor invasiveness of lung squamous cell carcinoma (LSCC). In two independent clinical cohorts from tissue microarrays (TMA: n = 208 patients) and whole sections (WS: n = 99 patients), TF was associated with poor prognosis and increased risk of blood vessel infiltration. A third cohort from The Cancer Genome Atlas (TCGA: n = 335 patients) confirmed the poor prognostic value of TF using a similar human-based score on hematoxylin-eosin staining. Integration of RNA-seq data from TCGA and LC-MS/MS proteomics from WS revealed an upregulation of extracellular matrix remodeling and focal adhesion processes in tumors with high TF, supporting their increased invasive potential. This proposed histologic parameter is an independent and unfavorable prognostic marker that could be established as a new grading parameter for LSCC. Cancer Res; 77(10); 2585–93. ©2017 AACR.