CD44 plays important roles in the tumor progression and has various isoforms, which are generated by the alternative splicing of CD44 mRNA. The mRNA of CD44 standard (CD44s) isoform is produced by constant region exons including the first five (1 to 5) and the last five (16 to 20). The mRNAs of CD44 variant (CD44v) isoform are produced by the assembling of variant exons (v1–v10) with the constant region exons of CD44s. CD44s and CD44v receive the post-translational modifications, such as N-glycosylation and O-glycosylation. Both CD44s and CD44v (pan-CD44) can attach to hyaluronic acid, which is important for cellular adhesion, homing, and motility. CD44v isoverexpressed in tumors and promotes tumor malignant progression through the binding to growth factors, and the acquisition of invasiveness, stemness, and drug resistance. These were mediated by the unique functions of the variant‘s exon-encoded region. Our group have established anti-CD44 monoclonal antibodies (mAbs) using the Cell-Based Immunization and Screening (CBIS) method. We would like to introduce the isoform-specific mAbs against CD44 and their application including flow cytometry and immunohistochemistry.