The sex-linked bleeding disorder hemophilia B is caused by defects in the protein Factor IX (hFIX). Clinically, hemophilia is the inability to form a fibrous clot upon injury and without prophylactic treatment can lead to internal bleeding in the joint spaces, causing hemophilic arthritis. Historically, hemophilia has been treated using blood transfusions; however in the past 20 years much research has gone into developing a gene therapy approach to transfer a functional copy of the gene to the either the skeletal muscle or liver in vivo or to stem cell populations ex vivo of affected individuals. Human clinical trials using a vector devised from the replication deficient parvovirus Adeno-associated virus (AAV) were problematic in that they elicited an immune response previously unseen in murine and canine models. Here, we used an immunogenic rabbit model to assess the transduction efficiency of two AAV serotypes (AAV5 and AAV6) in order to compare their relative success of transduction, initiation of an immune response, biodistribution, and kinetics of vector clearance from germline cells compared to the previously investigated serotypes of AAV2 and AAV8. The transduction success of AAV5 was limited in both hFIX expression and biodistribution while eliciting a large immune response to AAV capsid proteins. Conversely the AAV6 vector was capable of limiting the immune response to the capsid and achieve hFIX expression, with a limited antibody response to non-native hFIX, a downfall of both previous vector serotypes. AAV6 is a beneficial serotype for further investigation into a gene transfer therapy for hemophilia.