Our laboratory focuses on the molecular characterization of a non-pathogenic strain of fowl adenovirus (FAdV) 9 and its development as a versatile vaccine vector platform. The objectives of this study were to optimize transgene expression by recombinant viruses. High expression promoters and a post-transcriptional regulatory element were evaluated for their ability to improve expression of enhanced green fluorescent protein (EGFP) in recombinant FAdVs. These findings were compared to our current system that employs the human cytomegalovirus (CMV) promoter to express a transgene. EGFP expression was assessed by fluorometry and Western blots. A synthetic CMV enhancer/chicken β-actin (CAG) promoter and the human elongation factor 1 alpha (EF1α) promoter significantly increased expression of EGFP compared to the CMV promoter. However, expression was significantly decreased in the presence of woodchuck hepatitis virus post-transcriptional regulatory element (WPRE). The results provide novel insight into avian vaccine design and optimization of transgene expression by FAdV vectors.