Master differentiation transcription factors (MDFs) play decisive roles in cell lineage commitment. Paired box 5 (Pax5) is one of MDFs essential for differentiation of pre-B cells into mature B cells. Here, we analyzed the 5'-genomic structure and transcription of the chicken Pax5 (cPax5) gene in the chicken mature B cell line, DT40. We showed that the cPax5 gene has two first exons: exon 1A contains long AG repeats, while exon 1B has high GC contents. The exons 1A and 1B had one and three major transcription start sites, respectively. Semi-quantitative RT-PCR revealed that comparable amounts of mRNA are transcribed from the exons 1A and 1B. Interestingly, the transcription start site of the cPax5 exon 1A was chicken-specific. In addition, the cPax5 promoter upstream of the exon 1A had no homology with the human and mouse Pax5 promoters. Thus, the mechanisms regulating transcription of the Pax5 exon 1A might not be conserved among species. Furthermore, we determined the physical structure of the exons 1A, 1B, and 2 in the genome of DT40 cells. Our results will be useful for elucidating mechanisms that control cPax5 transcription and B cell lineage commitment, which is conserved or not conserved among different species.