Intratumor heterogeneity for HER2 in HER2-positive breast cancer is a driver of resistance to HER2-targeted therapies. The advancement of treatments for HER2 heterogeneous tumors has been hindered by the lack of preclinical models that accurately mimic the human disease. Here we describe human HER2 heterogeneous breast cancer models composed of ERBB2 amplified (HER2hi) and non-amplified (HER2lo) cell populations derived from the same tumor. Utilizing these models, together with cellular barcoding, we demonstrate subclonal cooperation between HER2hi and HER2lo subpopulations. Furthermore, HER2lo cells drive resistance to HER2-targeting antibody-drug conjugates (ADC) like T-DXd but are sensitive to HER2 kinase inhibitors. CRISPR screens in heterogeneous co-cultures identified sensitizers of HER2lo cells to T-DXd including ABCC1 and USP9X. USP9X inhibition enhances the lysosomal targeting of HER2, thereby potentiating ADC payload release and reducing tumor recurrence after T-DXd treatment. Our results elucidate the functional relevance of HER2 heterogeneity and propose improved therapies for these tumors.