<p>Background: Alternative splicing is an important mechanism that regulates gene expression and function in human cells. <em>OCT4</em>, a crucial pluripotency marker in embryonic stem/carcinoma cells generates several spliced variants in different cell types and cancers. The expression of <em>OCT4</em> in cancers has been challenged in many studies. The existence of several <em>OCT4</em> spliced variants and absence of specific discriminating primers is the main reason of this controversy. Therefore, using specific primers and discriminating <em>OCT4</em> variants from each other might help to reduce these discrepancies&nbsp; in carcinogenesis and stem cell researches.<br /> Methods: 17 various human cancer, pluripotent and normal cells were cultured and their RNAs were extracted. Related cDNAs were synthesized and the expression pattern of <em>OCT4</em> variants was investigated by RT-PCR assay. PCR products were cloned into pTZ57R/T vector and their authenticity was confirmed by DNA sequencing.<br /> Results: Expression pattern of <em>OCT4</em> variants (OCT4A, OCT4B and OCT4B1) was analyzed by RT-PCR assay and the authenticity of PCR products was confirmed by DNA sequencing. A novel spliced variant of OCT4 was discovered and named as OCT4B3. This variant was very similar to OCT4B2 transcript except that 207-nt of exon 1b is lost. Moreover, the expression pattern of OCT4B3 variant was investigated in 17 human cell types, where its expression was only found in astrocytoma and bladder cancer cell types 1321N1 and 5637, respectively.<br /> Conclusion: <em>OCT4</em> variants are differentially expressed in various human cancer cell lines. Moreover, a novel variant of <em>OCT4</em>, OCT4B3, was detected in two human cancer cell lines of bladder carcinoma (5637) and brain astrocytoma (1321N1) for the first time.</p>