CTCF, BORIS and YB-1 have been implicated in tumorgenesis. In this study, we examined the expressions of CTCF, BORIS and YB-1 in osteosarcoma (U2OS) and glioma (DBTRGO5MG) in comparison with their respective normal osteoblast (hFOB 1.19) and glial (SVGp12) cell lines. We examined any detected mutation for CTCF and YB1, and revealed the presence of physical in vivo CTCF~YB1 interaction. The level of CTCF mRNA was significantly higher in osteosarcoma compared to osteoblast cell line, while the level of YB-1 mRNA was similar in these cell lines. The expression of YB-1 mRNA was significantly elevated in glioma compared to glial cell line, whereas expression of CTCF mRNA was similar. The BORIS mRNA was detected only in osteosarcoma cell line. Nucleotides analysis showed undetected mutations at exon 4 and 5 (CTCF) and exon 7 (YB1) for both cancerous cell lines. Protein expression of CTCF revealed immunopositive for all cancerous and normal cell lines. YB- 1 protein expressions in glial , glioma and osteosarcoma cells was found to be immunopositive and anomalously migrated. We reported positive in vivo interaction of CTCF~YB1 protein in osteosarcoma and glioma cell lines. Different patterns of expression of CTCF,BORIS and YB- 1 mRNAs could indicate differential involvement of these transcriptional factors in tumorgenesis of osteosarcoma and glioma. Positive in vivo interaction of CTCF~YB1 in the osteosarcoma and glioma cell lines confirmed previous reports on both factors in tumor development.