Background: Gene CDKN2A, which encodes for p16INK4a/p14ARF is known to be important growth suppressor gene. CDKN2A gene alteration has been reported in non-small cell lung cancer (NSCLC). However, the demographic and clinical features of NSCLC with CDKN2A, coexisting gene alteration and association with immunotherapy biomarkers such as PD-L1 and tumor mutation burden are unknown. Methods: Tumor next-generation sequencing data from 197 NSCLC patients who are diagnosed at Mayo Clinic FL are retrospectively analyzed. Patients with CDKN2A gene alterations are identified. Data including demographic feature, clinical feature, coexisting gene alteration and association with immunotherapy biomarkers such as PD-L1 and tumor mutation burden are investigated. Results: One hundred ninety-seven patients with NSCLC were identified, and 49 (24.8%) had CDKN2A gene alteration, including 32 gene loss, 4 somatic mutations and 3 deletions. The medium age of patients with CDKN2A gene alteration was 66.7. It was slightly more predominant in male than female (53.1% versus 46.9%) and more in smoker than never-smoker (65.3% versus 34.7%). Among patients with CDKN2A gene alteration, 83.7% (41/49) were found in lung adenocarcinoma, 10.2% were found in lung squamous cell carcinoma and 6.1% were found in other histology. The most common coexisting gene alterations associated with CDKN2A are CDKN2B (61%) followed by TP53 (51%), KRAS (35%), EGFR (14.2%), STK11 (12.2%), MET (10%), PI3KCA (10%), HER-2 (8%), ALK (4%), BRAF (2%). Interestingly, 65% of patients with CDKN2A gene alteration were found to have expression of PD-L1 (defined by > 1% PD-L1). Among them, 61.5% patients have high expression of PD-L1 (defined by > 50% PD-L1). Patients with CDKN2A gene alteration are also associated with high tumor mutation burden (8.77mutation/mb). Conclusions: CDKN2A gene alteration is commonly seen in NSCLC. Co-existing driver mutations such as EGFR, MET, HER-2, ALK, BRAF are found. Immunotherapy related biomarkers such as high expression level of PD-L1 and tumor mutation burden are found common in patients with CDKN2A gene alteration, indicating the likelihood of clinical benefit to immunotherapy and warrant further investigation in a larger, prospective study.