Sacituzumab govitecan (SG), the first antibody-drug conjugate (ADC) approved for triple-negative breast cancer, incorporates the anti-TROP2 antibody hRS7 conjugated to a topoisomerase-1 (TOP1) inhibitor payload. We sought to identify mechanisms of SG resistance through RNA and whole-exome sequencing of pretreatment and postprogression specimens. One patient exhibiting de novo progression lacked TROP2 expression, in contrast to robust TROP2 expression and focal genomic amplification of TACSTD2/TROP2 observed in a patient with a deep, prolonged response to SG. Analysis of acquired genomic resistance in this case revealed one phylogenetic branch harboring a canonical TOP1E418K resistance mutation and subsequent frameshift TOP1 mutation, whereas a distinct branch exhibited a novel TACSTD2/TROP2T256R missense mutation. Reconstitution experiments demonstrated that TROP2T256R confers SG resistance via defective plasma membrane localization and reduced cell-surface binding by hRS7. These findings highlight parallel genomic alterations in both antibody and payload targets associated with resistance to SG. Significanc e: These findings underscore TROP2 as a response determinant and reveal acquired SG resistance mechanisms involving the direct antibody and drug payload targets in distinct metastatic subclones of an individual patient. This study highlights the specificity of SG and illustrates how such mechanisms will inform therapeutic strategies to overcome ADC resistance.