Background: Multiple large-scale tumor genomic profiling efforts have been undertaken in osteosarcoma, however little is known about the spatial and temporal intratumor heterogeneity and how it may drive treatment resistance. Methods: We performed 30-80x whole genome sequencing (WGS) of 37 tumor samples from 8 patients with relapsed or refractory osteosarcoma. A set of high confidence single nucleotide variants (SNV), copy number alterations (CNA), structural variations (SV) were called for each sample using our pediatric expanded genomics pipeline and an evolutionary analysis was performed using a custom pipeline of computational tools. Results: Of the 8 patients in our cohort, 4 had localized disease at diagnosis (OSCE4, OSCE5, OSCE6, OSCE9) and 4 had metastatic disease at diagnosis (OSCE1, OSCE2, OSCE3, OSCE10). There were 17 samples from primary sites, 7 were pretreatment biopsies, 10 from on therapy primary resections. 20 samples came from metastatic sites, 15 of which were from lung metastases. Driver gene SNV’s were identified in 5 of 8 patients, including TP53 (OSCE1), ATRX (OSCE3, OSCE10), RB1 (OSCE4), and CDKN2A (OSCE9). No new driver SNV’s emerged post-therapy in any patient. HATCHet, an algorithm which infers clone specific copy number alterations, identified subclonal CNAs in all but one patient (OSCE2). In the 7 patients with subclonal CNAs, 6 had two copy number clones identified, and 1 patient (OSCE10) had three copy number clones identified. In 5 patients (OSCE1, OSCE4, OSCE5, OSCE6, OSCE10) there is a copy number clone that is subclonal in the primary tumor which emerges and dominates at subsequent relapses. The resistant clone in each of these cases had either MYC gain/amplification. Amplifications in CCNE1 (OSCE1), RAD21 (OSCE4, OSCE5, OSCE10), VEGFA (OSCE1, OSCE9), IGF1R (OSCE6) were also identified as potential drivers in the resistant copy number clones. In two of these patients (OSCE1, OSCE6), the treatment resistant subclone becomes the dominant copy number clone by the time of primary resection. SNV based phylogenies revealed monoclonal and polyclonal seeding of metastases and monophyletic and polyphyletic modes of dissemination. Over half the new mutations acquired in recurrent disease were attributed to HRD or cisplatin mutational signatures. Conclusions: Subclonal copy number clones emerge and dominate in relapsed osteosarcoma, with MYC gain/amplification a defining characteristic in our cohort. Selective pressure from neoadjuvant chemotherapy reveals this clone at the time of primary resection, implying genomic profiling at this timepoint may be more reflective of its metastatic potential.