Arrangement and density of collagen fibers in burn scar were detected by second harmonic and polarized light imaging techniques in mice. The burn model was established on the back of mice, excisions of burn wounds were undertaken following current clinical practice, arrangement and density of collagen fibers in papillary layer and reticular layer of scar and scar edge were studied by second harmonic and polarized light imaging techniques in vivo and ex vivo. The results of second harmonic imaging showed that collagen fibers were randomly arranged, arrangement consistency was low, collagen fibers were sparse, area density was small in papillary layer and reticular layer of normal skin, however, arrangement consistency and area density were increased significantly in papillary layer and reticular layer of scar and scar edge. The results of liquid crystal polarized light imaging technology showed that variation of azimuth of collagen fibers were reduced significantly, retardance were increased in papillary layer and reticular layer of scar and scar edge compared with normal skin. We found that arrangement and density of collagen fibers in papillary layer and reticular layer of scar and scar edge were changed by second harmonic and polarized light imaging technology, the results of second harmonic and liquid crystal polarized light imaging technology had the same trend. The results would lay the foundation for non-invasive identification of scar tissue through second harmonic imaging technology in future, and provide methods for diagnosing scars in human and assess effectiveness of scar treatment.