Animal models have been critical to the development of novel therapeutics in hemophilia. A deficiency of current murine models of hemophilia B is that they are all due to gene deletions, a type of mutation that is relatively rare in the human hemophilia population. We generated mice with a range of mutations in the Factor IX (F.IX) gene; these more faithfully reflect the types of mutations that cause disease in the human population. Transgenic mice expressing either wild-type human F.IX (hF.IX), or F.IX variants with premature translation termination codons, or missense mutations, under the control of the murine transthyretin promoter, were generated and crossed with mice carrying a large deletion of the murine F.IX gene. Gene copy number, F.IX transcript levels in the liver, intrahepatocyte protein expression, and circulating levels of F.IX protein in the mice were determined and compared with data generated by transient transfection assays using the same F.IX variants. Mice were injected with a viral vector expressing hF.IX and displayed a range of immune responses to the transgene product, depending on the underlying mutation. These new mouse models faithfully mimic the mutations causing human disease, and will prove useful for testing novel therapies for hemophilia.