Targeting HER2 is effective in 24% of ERBB2 amplified metastatic colorectal cancer; however, secondary resistance occurs in most of the cases. We studied the evolution of individual metastases during treatment to discover spatially resolved determinants of resistance. Circulating tumor DNA (ctDNA) analysis identified alterations associated with resistance in the majority of refractory patients. ctDNA profiles and lesion-specific radiographic reports revealed organ- or metastasis-private evolutionary patterns. When radiologic assessments documented progressive disease in target lesions, response to HER2 blockade was retained in other metastases. Genomic and functional analyses on samples and cell models from eight metastases of a patient co-recruited to a postmortem study unveiled lesion-specific evolutionary trees and pharmacologic vulnerabilities. Lesion size and contribution of distinct metastases to plasma ctDNA were correlated. Display omitted •ERBB2, RAS, PIK3CA mutations are associated with resistance to HER2 blockade in mCRC•A liquid biopsy test would have identified >85% primary resistance cases•Lesion size and contribution to plasma ctDNA were correlated•Patterns of lesion-specific mutations and TCR were longitudinally compared in blood Siravegna et al. identify genetic events associated with ERBB2 amplified metastatic colorectal cancers resistant to trastuzumab plus lapatinib treatment and reveal lesion-private evolutionary patterns. Analyses of metastases from a patient unveil metastasis-specific evolution and pharmacologic vulnerabilities.