Adeno-associated viruses (AAVs) are widely present in human tissues, with 18% of tissues showing endogenous AAV sequences, primarily in the liver and bone marrow. AAVs are members of the Dependovirus genus within the Parvoviridae family and have potential as gene therapy vectors. This study identified diverse AAV clades, including a hybrid clade of two serotypes and a clade found in humans and nonhuman primates, suggesting cross-species transmission. AAVs were detected in various tissues, with the liver and spleen being the most common sites. The study characterized AAV sequences from human and nonhuman primate tissues, revealing 67 human and 86 primate AAV clones. Phylogenetic analysis showed that AAVs form distinct clades, with some clades containing hybrid sequences. The AAV2-AAV3 hybrid clade was identified, and AAV8 was found in both humans and nonhuman primates. Serologic and functional analyses indicated that AAVs have distinct properties, with some clades showing cross-reactivity. The study also evaluated the transduction efficiency of AAV vectors in vitro and in vivo, showing varying efficiencies depending on the AAV serotype. The findings suggest that AAVs are widely disseminated in humans and nonhuman primates, with potential implications for gene therapy and viral transmission. The study highlights the importance of understanding AAV biology for the safe and effective use of AAV as gene therapy vectors.Adeno-associated viruses (AAVs) are widely present in human tissues, with 18% of tissues showing endogenous AAV sequences, primarily in the liver and bone marrow. AAVs are members of the Dependovirus genus within the Parvoviridae family and have potential as gene therapy vectors. This study identified diverse AAV clades, including a hybrid clade of two serotypes and a clade found in humans and nonhuman primates, suggesting cross-species transmission. AAVs were detected in various tissues, with the liver and spleen being the most common sites. The study characterized AAV sequences from human and nonhuman primate tissues, revealing 67 human and 86 primate AAV clones. Phylogenetic analysis showed that AAVs form distinct clades, with some clades containing hybrid sequences. The AAV2-AAV3 hybrid clade was identified, and AAV8 was found in both humans and nonhuman primates. Serologic and functional analyses indicated that AAVs have distinct properties, with some clades showing cross-reactivity. The study also evaluated the transduction efficiency of AAV vectors in vitro and in vivo, showing varying efficiencies depending on the AAV serotype. The findings suggest that AAVs are widely disseminated in humans and nonhuman primates, with potential implications for gene therapy and viral transmission. The study highlights the importance of understanding AAV biology for the safe and effective use of AAV as gene therapy vectors.