CD44 has been known as a marker of tumor-initiating cells, and plays pro-tumorigenic functions in many cancers. The splicing variants play critical roles in the malignant progression of cancers by promoting stemness, cancer cell invasion or metastasis, and resistance to chemo- and radiotherapy. To understand each CD44 variant (CD44v) function is essential to know the property of cancers and the establishment of the therapy. However, the function of the variant 4-encoded region has not been elucidated. Therefore, specific monoclonal antibodies (mAbs) against variant 4 are indispensable for basic research, tumor diagnosis, and therapy. In this study, we established anti-CD44 variant 4 (CD44v4) mAbs by immunizing mice with a peptide containing the variant 4-encoded region. We next performed flow cytometry, western blotting, and immunohistochemistry to characterize them. One of the established clones (C44Mab-108; IgG1, kappa) reacted with CD44v3-10-overexpressed Chinese hamster ovary-K1 cells (CHO/CD44v3-10). The KD of C44Mab-108 for CHO/CD44 v3-10 was 3.4 × 10-7 M. In western blot analysis, C44Mab-108 detected CD44v3-10 in the lysate of CHO/CD44v3-10 cells. Furthermore, C44Mab-108 stained formalin-fixed paraffin-embedded (FFPE) oral squamous carcinoma tissues in immunohistochemistry. These results indicated that C44Mab-108 is useful to detect CD44v4 in immunohistochemistry using FFPE tissues.