Gene therapy holds considerable promise for treatment of musculoskeletal and neuromuscular disorders. However, low muscle affinity in natural adeno-associated virus (AAV) vectors require high viral doses for systemic delivery, which have been associated with adverse events across clinical trials. Thus, there is a need to identify novel vectors with enhanced muscle targeting to improve gene delivery efficiency with lower doses. Through iterative rounds of selection using an RGD peptide–inserted AAV library in cynomolgus macaques, we identified AAV capsid variants with improved muscle transduction. We validated three top candidates which showed good species cross-reactivity, BIIB-Cap004, BIIB-Cap011 and BIIB-Cap018 in mouse and human muscle cells, and NHP using a reporter cargo. We performed single-nuclei RNAseq in mice to identify the muscle cell populations with the highest transduction. Additionally, we used a micro-dystrophin cargo using these novel RGD capsids to characterize functional recovery in human DMD muscle cells (2D differentiated skeletal myotubes, 3D myobundles, and iPSC-cardiomyocytes) and the DMD mdx mouse model. Here we demonstrate these novel muscle-tropic capsids have higher potency than AAVrh74 and AAV9, which could potentially drive the development of safer gene therapies targeting muscle diseases.