Self-complementary adeno-associated virus (scAAV) vectors can significantly minimize the vector load required to achieve sustained transgene expression. In this study, transcriptional regulatory elements were systematically screened to produce constitutive and liver-specific scAAV factor IX (FIX) expression cassettes. In addition, optimization of GC content, cis- regulatory elements, and codon usage in the human FIX (hFIX) transgene increased expression 4–20-fold. A vector was developed that was capable of expressing high FIX levels in comparison with the single-stranded (ss) AAV vector used in a recent clinical trial. The ssAAV and scAAV vectors display different transgene expression and genome stability patterns in the liver, as determined by immunohistochemical staining, in situ messenger RNA (mRNA) hybridization and vector genome quantitation. The ssAAV2 vector promoted strong FIX expression in only a subset of hepatocytes. The scAAV2-hFIX vector showed widespread (∼80% of hepatocytes), moderate FIX expression levels similar to normal livers with correction of coagulation function in FIX-deficient mice. The ability of low dose scAAV-FIX vectors to achieve near-physiological expression may circumvent inflammatory responses in the liver. In addition to providing an improved scAAV vector for potential application in future hemophilia B clinical trials and liver-directed gene delivery, these studies underscore the need for rigorous analysis and optimization of vector genome cassettes.