Gastrointestinal stromal tumors (GISTs) are the most common mesenchymal tumors arising in the digest tract. It brings a challenge to diagnosis because it is asymptomatic clinically. It is well known that tumor development is often accompanied by the changes in the morphology of collagen fibers. Nowadays, an emerging optical imaging technique, second-harmonic generation (SHG), can directly identify collagen fibers without staining due to its noncentrosymmetric properties. Therefore, in this study, we attempt to assess the feasibility of SHG imaging for detecting GISTs by monitoring the morphological changes of collagen fibers in tumor microenvironment. We found that collagen alterations occurred obviously in the GISTs by comparing with normal tissues, and furthermore, two morphological features from SHG images were extracted to quantitatively assess the morphological difference of collagen fibers between normal muscular layer and GISTs by means of automated image analysis. Quantitative analyses show a significant difference in the two collagen features. This study demonstrates the potential of SHG imaging as an adjunctive diagnostic tool for label-free identification of GISTs.