Background: The number of multiple primary lung cancer (MPLC) patients has rapidly increased in recent years. However, germline mutations and information regarding the etiology of MPLC is very rare. In this study, we sought to determine the prevalence and clinical significance of germline pathogenic/likely pathogenic (P/LP) variants in patients with MPLC. Methods: A total of 111 MPLC patients undergoing radical surgery were enrolled in this study and 625 patients with single-focus lung cancer (SFLC) were analyzed as a control. Tumor and blood lymphocyte DNA from patients were sequenced using next-generation sequencing (NGS) platform interrogating up to 808 cancer-associated genes. Interpretation of sequence variations was classified according to the American College of Medical Genetics and Genomics guidelines. Results: A total of 15 germline P/LP mutations in 12.6% (14/111) of MPLC patients were observed, including one patient with 2 germline alterations; of these, 9 patients (64.3%) had germline mutations in DNA-damage repair (DDR) pathway genes in which the most commonly genes were WRN (14.3%, 2/14) and BRCA1/2 (14.3%, 2/14), followed by RAD50 and MSH3. Similarly, 68 germline mutations were identified in 67 (10.7%)SFLCs, and 54 (79.4%) mutation genes were DDR genes including RAD50 (10.4%, 7/67) showed the highest frequency. The somatic mutations of EGFR (59.5% vs. 48.5%) were enriched in P/LP alterations carriers, while TP53 (12.4% vs. 4.8%) was more common in MPLC patients without P/LP alterations. Besides, lower BRAF and MET mutation frequency were observed in SPLC with germline alterations (p < 0.05), compared to those who did not. MPLC were more likely to be patients with family history of cancers (32.4% vs. 25.0%, p = 0.099) than SFLC, while the proportion of patients with family history of cancers found no significant difference between P/LP and non-P/LP groups. MPLC patients with germline P/LP variances were diagnosed at younger age ( < 40 years old) compared to non-carriers. For MPLC, the ratio of males is not associated with P/LP variations. However, significantly higher proportion of germline P/LP mutations was detected in males in SFLC (61.2% vs. 48.4%, p = 0.048). Patients with a history of smoking were significantly less enriched in MPLC (p = 0.030) compared to SPLC but were independent of whether harbored germline mutations. Conclusions: Taken together, ur results show that in the first large Chinese cohort of MPLC, 12.6% of patients carry germline variants. In addition, a more detailed analysis of the spectrum of germline mutations and clinical characteristics of MPLC may provide clues for the oncology treatment strategy and cancer prevention for MPLC.