Background: Non-small cell lung cancer (NSCLC) is the most common subtype of lung cancer and leading cause of cancer related death worldwide. Acquired genetic alterations in major driver genes including EGFR, KRAS, NRAS, BRAF, ALK and ROS1 are the most common mutations in NSCLC and certain mutations are associated with drug sensitivity or resistance. Hence, the mutation profiles of NSCLC patients are vital to guide targeted therapy and monitor the tumor recurrence, thereby improving the survival rate. The latest Globocan data showed that lung cancer ranks as the second most common cancer in Vietnam with high incidence and mortality rate. Nonetheless, the mutation spectrum of Vietnamese NSCLC patients has not been profiled thoroughly and current views on mutation testing largely rely on data obtained from previous prospective studies in Caucasian or East Asian cohorts. Methods: Massive parallel sequencing was employed to detect both somatic point mutations and rearrangement in six major driver genes in tissue biopsies from 350 NSCLC patients in Vietnam. The χ2/Fisher’s exact test was performed to compare mutation frequency between different cohorts. Additionally, both univariate and multivariate tests were used to identify clinical factors associated with mutation prevalence. Results:EGFR (32.3%) and KRAS (20%) accounted for the most frequently mutated genes, followed by ALK (5.4%), ROS1 (2.9%), BRAF (1.1%) and NRAS (0.6%). Our data showed a unique pattern of mutation profiles in Vietnamese NSCLC patients, with significant enrichment of KRAS mutation as compared to reported East Asian cohorts (20% versus 8%-10%, p < 0.05). Furthermore, EGFR and KRAS mutation frequencies were significantly associated with patients’ gender, with EGFR mutations more commonly detected in female than in male (48.1% versus 26.9%, p < 0.00001) while higher KRAS mutation frequency in male than in female (30.7% versus 9.2%, p < 0.0001). Young patients aged below the median age of 61 years had significantly higher tendency to acquire rearrangements in ALK (p = 0.02) and ROS1 (p = 0.03) than elderly patients. Conclusions: To our knowledge, our study is the first to reveal the mutation profiles of major druggable genes in a large cohort of Vietnamese NSCLC patients.