We report the generation of a new class of adeno-associated virus serotype 9 (AAV9)-derived vectors displaying selective loss of liver tropism and demonstrating potential for cardiac and musculoskeletal gene transfer applications. Random mutagenesis of residues within a surface-exposed region of the major AAV9 capsid protein yielded a capsid library with mutations clustered at the icosahedral threefold symmetry axis. Using a combination of sequence analysis, structural models, and in vivo screening, we identified several functionally diverse AAV9 variants. The latter were classified into three functional subgroups, with respect to parental AAV9 displaying: (i) decreased transduction efficiency across multiple tissues; (ii) a selective decrease in liver transduction, or (iii) a similar transduction profile. Notably, variants 9.45 and 9.61 (subgroup II) displayed 10- to 25-fold lower gene transfer efficiency in liver, while transducing cardiac and skeletal muscle as efficiently as AAV9. These results were further corroborated by quantitation of vector genome copies and histological analysis of reporter (tdTomato) gene expression. The study highlights the feasibility of generating AAV vectors with selectively ablated tissue tropism, which when combined with other targeting strategies could allow sharply segregated gene expression. Liver-detargeted AAV9 variants described herein are excellent candidates for preclinical evaluation in animal models of cardiac and musculoskeletal disease.