INTRODUCTION: Homeobox genes are highly conserved and play critical roles in brain development. Recently, we have found that mammals have an additional fragment of approximately 20 amino acids in Emx1 and a poly-(AL)6-7 in Emx2, compared to other amniotes. It has been shown that Emx1 and Emx2 have synergistic actions in the brain development. These reports raise an interesting issue whether the differences of Emx1 and Emx2 between mammals and non-mammals are concerned with the organization and evolution of amniote pallium. METHODS: Lentiviruses expressing mouse Emx1 and Emx2 (mEmx1/2) with additional fragments were injected into the ventricle of the chick telencephalon at embryonic day 3 to study the effects of mEmx1/2 on the development of chick pallium, whereas injections of lentiviruses containing chick Emx1 and Emx2 (cEmx1/2), no targeted gene insert or saline were as controls. The expressions of reelin, vimentin, GABA and MAP2, neurogenesis patterns for calbindin (CB) and parvalbumin (PV) neurons and the sizes of anterior commissure (AC) were then studied by immuohistochemical staining, and open-field tests were performed to assess locomotor activities and curious or exploratory behaviors of the chicks. RESULTS: Following the injections of lentiviruses expressing mEmx1/2, the expressions of reelin, vimentin, GABA, and MAP2 increased in most parts of Wulst (W) and mesopallium (M), but not most of nidopallium (N). Neurogenesis patterns for CB and PV neurons changed toward mammalian inside-out one, and the sizes of AC staining for neurofilament were significantly larger. In addition, post-hatchling chicks showed higher rates of passive avoidance after training, but no significant differences in the total distance traveled and the percentage of time spent in the central rectangle, compared to those in the control groups. CONCLUSION: The present study indicated that mEmx1/2 had effects on the development of chick pallium, suggesting that they are probably involved in the organization and evolution of amniote pallium.