Purpose: Non-small cell lung cancer (NSCLC) is the most common cause of cancer-related deaths worldwide. There is an unmet need to develop novel clinically relevant models of NSCLC to accelerate identification of drug targets and our understanding of the disease. Experimental Design: Thirty surgically resected NSCLC primary patient tissue and 35 previously established patient-derived xenograft (PDX) models were processed for organoid culture establishment. Organoids were histologically and molecularly characterized by cytology and histology, exome sequencing, and RNA-sequencing analysis. Tumorigenicity was assessed through subcutaneous injection of organoids in NOD/SCID mice. Organoids were subjected to drug testing using EGFR, FGFR, and MEK-targeted therapies. Results: We have identified cell culture conditions favoring the establishment of short-term and long-term expansion of NSCLC organoids derived from primary lung patient and PDX tumor tissue. The NSCLC organoids recapitulated the histology of the patient and PDX tumor. They also retained tumorigenicity, as evidenced by cytologic features of malignancy, xenograft formation, preservation of mutations, copy number aberrations, and gene expression profiles between the organoid and matched parental tumor tissue by whole-exome and RNA sequencing. NSCLC organoid models also preserved the sensitivity of the matched parental tumor to targeted therapeutics, and could be used to validate or discover biomarker-drug combinations. Conclusions: Our panel of NSCLC organoids closely recapitulates the genomics and biology of patient tumors, and is a potential platform for drug testing and biomarker validation.