Background: Despite improvements in survival with immune checkpoint inhibition (ICI), the majority of patients develop acquired resistance to ICI after an initial benefit. However, the mechanisms underlying acquired resistance to ICI in NSCLC are largely unknown. Methods: Patients with advanced NSCLC treated with ICI at the Dana-Farber Cancer Institute (DFCI), and whose tumors underwent genomic profiling before and after ICI, with no intervening therapies, were included. Mutations, tumor mutational burden (TMB), copy number variations (CNVs), and PD-L1 tumor proportion score (TPS) were compared between pre- and post-ICI samples. Acquired resistance was defined as the development of disease progression after an initial objective response, or stable disease ≥3 months with PD-(L)1 blockade. Results: Among 1763 patients with advanced NSCLC who received ICI, 45 had matched pre- and post-ICI tissue samples available for genomic profiling. Putative mechanisms of resistance were identified in 55% of cases (N = 25). Five patients (20%) acquired an STK11 mutation, one patient (4%) acquired a KEAP1 mutation, and another patient (4%) developed concurrent KEAP1 and SMARCA4 mutations. A patient (4%) with KRAS G12C-mutant NSCLC developed concurrent STK11 and KEAP1 mutations at resistance. In 3 cases (12%) with pre-existing STK11 or KEAP1 mutations prior to ICI administration, we identified acquired copy losses of STK11 and KEAP1, respectively, resulting in bi-allelic inactivation of these genes. Acquired beta-2-microglobulin (B2M) mutations were detected in 3 patients (12%), one of whom developed concurrent B2M copy loss, indicating bi-allelic inactivation. Eight additional patients (32%) developed B2M gene deletions. Other acquired alterations that have been implicated in ICI resistance included CDKN2A/B loss (N = 10, 40%), including 5 with bi-allelic deletion, acquired PTEN deletions (N = 5, 20%), and MDM2 amplification (N = 2, 8%). When we examined alterations in immune checkpoint genes, we identified acquired CD274 (PD-L1) and PDCD1LG2 (PD-L2) loss in 8% of cases (N = 2), and bi-allelic deletion in one case (4%). Intervening ICI did not affect TMB (median TMB: 8.7 [pre-ICI] vs 9.1 [post-ICI] mut/Mb, P = 0.6), PD-L1 expression (median PD-L1 TPS: 3% [pre-ICI] vs 5.0% [post-ICI] mut/Mb, P = 0.5), or aneuploidy levels (as fraction of genome altered [FGA]) (median FGA: 18.4% [pre-ICI] vs 21.1% [post-ICI], P = 0.2), indicating that acquired gene level CNVs were not a reflection of increased cancer aneuploidy. In a control cohort of 30 patients with pre- and post-chemotherapy matched samples which underwent genomic profiling, no acquired mutations in STK11, KEAP1, SMARCA4, or B2M were detected. Conclusions: Mechanisms of acquired resistance to PD-(L)1 blockade are heterogenous, and new therapeutic strategies are required to delay and overcome ICI resistance in patients with NSCLC.