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Abstract: In the present study, we cloned and characterized the buffalo (Bubalus bubalis) OCT4 ortholog expressed in embryonic stem cell (ESC) like cells and its promoter region. The 5′- and 3′-RACE experiments were conducted to analyze the transcription initiation site and regulatory regions. The comparative analysis of buffalo OCT4 promoter with other mammalian orthologs revealed high conservation. Among the regulatory regions highest similarity was observed between buffalo, bovine and sheep. Interestingly, buffalo OCT4 promoter exhibited a 78bp deletion between two proximal enhancers (PE-1A and PE-1B) when compared to other mammalian orthologs. 5′-RACE revealed four different transcription start sites for OCT4 gene. As far as we know there is no previous report regarding multiple transcription initiation sites for OCT4 gene in any species. In addition, we identified expression of four pseudogenes in buffalo ESC-like cells. Among the multiple transcripts characterized, we found four cDNA clones (1083bp) derived from ESC-like cells sharing 96.9–99.3% sequence homology with the parent gene and having the capacity of encoding 139, 206, 206 and 324 amino acid long truncated proteins. Multiple pseudogenes have been proposed for OCT4 which might contribute to the false detection of this gene during expression studies. However, only few of them were reported to be transcribed and none were reported to be translated in stem cells. Western blot analysis of OCT4 protein using ESC-like cells revealed multiple bands, indicating that some of the hypothetical pseudogenes are being translated. These novel pseudogenes or their protein products may have some important regulatory functions. [Copyright &y& Elsevier]