In this work we demonstrated the successful production of transgenic chickens expressing the enhanced green fluorescence protein (EGFP) gene. Replication-defective recombinant retroviruses produced from vesicular stomatitis virus G glycoprotein pseudotyped retrovirus vector system were injected beneath the blastoderm of non-incubated chicken embryos (stage X). From 129 injected eggs, 13 chicks hatched after 21 days of incubation. All hatched chicks were found to express vector-encoded EGFP gene, which was under the control of the Rous sarcoma virus promoter and boosted post-transcriptionally by woodchuck hepatitis virus post-transcriptional regulatory element sequence. Green fluorescent signals, indicative of the EGFP gene expression, were detected in various body parts, including head, limb, eye, toe, and several internal organs. Genomic incorporation of the transgene was also proven by Southern blot assay. Our results show the exceptional versatile effectiveness of the EGFP gene as a marker in the gene expression-related studies which therefore would be very helpful in establishing a useful transgenic chicken model system for studies on embryo development and for efficient production of transgenic chickens as bioreactors.