Background: Osimertinib is a standard drug for first-line treatment of patients with advanced non–small cell lung cancer (NSCLC) harboring EGFR mutations (mt). While tumor mutational burden (TMB)-high and co-occurring genetic alterations (alt) have been reported to be negatively associated with the efficacy of other EGFR-TKIs, the impact of co-occurring genetic alt with EGFR major mt on the efficacy of osimertinib remains unclear. Methods: In a multi-institutional genomic screening project (LC-SCRUM-Asia), we have analyzed lung cancer patients for genomic alt by a targeted next-generation sequencing (NGS) system, Oncomine Comprehensive Assay and Genexus/OPA. We retrospectively evaluated the association between the genomic profile and efficacy of first-line osimertinib for EGFR-mutated NSCLC based on the LC-SCRUM-Asia database. Results: Between March 2015 and January 2022, 12,705 NSCLC patients were enrolled in the LC-SCRUM-Asia database, and EGFR mt was detected in 2,232 patients. Of these, 324 patients, including 171 with ex19del (53%) and 153 with L858R (47%), received first-line treatment with osimertinib. The patient characteristics were as follows: median age, 69 years (range 31-97); females, 64%; never-smokers, 57%; adenocarcinoma, 97%; and performance status 0-1, 99%. The frequency of compound EGFR mt and TMB were higher in the L858R (LR) group than in the ex19del (Ex19) group (compound mt (%), 12 vs. 4; mean TMB (mt/Mb), 3.4 vs. 2.5). There were no differences in the frequencies of other co-occurring genetic alt between the two groups. Higher TMB, alt of genes encoding receptor tyrosine kinase (RTK), including FGFR1, RET, MET etc., and amp of cell-cycle related genes were significantly associated with shorter progression-free survival (PFS) in the entire group (median PFS: TMB > 3 vs. ≤3 mt/Mb = 11.4 vs. 17.1 months; p = 0.023; RTK gene alt+ vs. alt- = 9.7 vs. 15.2 months; p = 0.014; cell-cycle gene amp+ vs. amp- = 10.6 vs. 15.6 months, p = 0.001). EGFR subgroup analysis showed that a higher TMB was significantly associated with a shorter PFS in the LR group (> 3 vs. ≤3 mt/Mb = 10.0 vs. 17.1 months, p < 0.001), but not in the Ex19 group. On the other hand, alt of genes encoding RTK and amp of cell-cycle related genes were significantly associated with a shorter PFS in the Ex19 group (RTK gene alt+ vs. alt- = 8.4 vs. 17.8 months, p = 0.008; cell-cycle gene amp+ vs. amp- = 10.6 vs. 17.5 months, p = 0.003), but not in the LR group. Multivariate analysis identified RTK gene alt in the Ex19 group and higher TMB in the LR group as being independently associated with a shorter PFS. Conclusions: First-line osimertinib treatment was less effective in NSCLC patients harboring Ex19 with other RTK gene alt or LR with a higher TMB, indicating that co-occurring genetic alt affecting the efficacy of osimertinib differ between NSCLC patients harboring Ex19 and LR.