Gene therapy using recombinant Adeno-associated viruses (AAV) is highly promising, but the presence of neutralizing antibodies against the AAV capsid proteins in humans limits its efficacy. Interestingly, clinical evidence exists that the AAV5 serotype can mediate gene transfer even in patients seropositive for AAV5. This makes AAV5 a very attractive template for AAV capsid diversification and molecular evolution by DNA shuffling, a process based on the fragmentation and homology-directed recombination of multiple parental capsid sequences. Alas, AAV5 shuffling is inefficient due to its low identity of <65% with all other primate AAV serotypes. One solution validated by us and others before is to codon-adapt the AAV5 capsid cDNA to the other parental AAV variants in a shuffled library. Here, we introduce an original and complementary improvement whose hallmark is the use of progressive DNA shuffling of AAV5 capsid sequences with other serotypes, specifically, wild-types AAV1, AAV2, AAV7, AAV9, or AAVrh10 (~65-73% homology with wild-type AAV5). Our new method can yield AAV capsid libraries with ~88-100% of individual clones containing at least one fragment from AAV5. Moreover, the mean AAV5 capsid sequence coverage per clone can surpass 35%. The benefits of our new AAV5 shuffling strategy over the conventional protocol will be exemplified in more detail with data on the diversity of representative AAV5-containing plasmid and virus libraries. By improving the shuffling of largely heterologous AAV variants such as AAV5, our advanced protocol facilitates the creation and selection of highly chimeric novel AAV capsids with unique properties for human gene transfer.