The study of gene function in vivo is considered one of the top achievements of modern biology, inasmuch as it provides tools to study gene function in the context of the whole animal. In chickens, techniques of DNA-mediated gene transfer are less advanced than in other animal or livestock models, and remain a significant challenge. The study presented here is the first to show that a hydrodynamics-based gene-transfer technique, originally developed for naked DNA transfer in mice, can be applied to chickens. Rapid injection of naked plasmids containing expression cassettes into the jugular vein of 6- to 10-day-old chicks resulted in specific expression of the transgenes. A CMV promoter-driven luciferase reporter gene was expressed at significant levels in the liver during the first 3 days post-injection with lower levels also detected in the kidney. Significantly, all injected birds showed detectable levels of luciferase expression. Similarly, injection of a plasmid containing the secreted human coagulation factor IX (hFIX) gene under the control of human alpha-1-anti-trypsin promoter resulted in detectable levels of the hFIX in the plasma during the first 2 days post-injection. The method described herein has the potential for a quick and simple route for gain and loss-of function experiments in chicken liver and kidney, as well as for studying systemic effects of secreted proteins and hormones.