Non-small cell lung cancer (NSCLC) often metastasizes to the brain, but identifying which patients will develop brain metastases (BM) is difficult. Macroautophagy/autophagy is critical for cancer initiation and progression. We hypothesized that genetic variants of autophagy-related genes may affect brain metastases (BM) in NSCLC patients. We genotyped 16 single nucleotide polymorphisms (SNPs) in 7 autophagy-related (ATG) genes (ATG3, ATG5, ATG7, ATG10, ATG12, ATG16L1, and MAP1LC3/LC3) by using DNA from blood samples of 323 NSCLC patients. Further, we evaluated the potential associations of these genes with subsequent BM development. Lung cancer cell lines stably transfected with ATG16L1: rs2241880 (T300A) were established. Mouse models of brain metastasis were developed using cells transfected with ATG16L1-300T or ATG16L1-300A. ATG10: rs10036653 and ATG16L1: rs2241880 were significantly associated with a decreased risk of BM (respective hazard ratios [HRs]=0.596, 95% confidence interval [CI] 0.398-0.894, P = 0.012; and HR = 0. 655, 95% CI 0.438-0.978, P = 0.039, respectively). ATG12: rs26532 was significantly associated with an increased risk of BM (HR = 1.644, 95% CI 1.049-2.576, P = 0.030). Invasion and migration assays indicated that transfection with ATG16L1-300T (vs. 300A) stimulated the migration of A549 cells. An in vivo metastasis assay revealed that transfection with ATG16L1-300T (vs. 300A) significantly increased brain metastasis. Our results indicate that genetic variations in autophagy-related genes can predict BM and that genome analysis would facilitate stratification of patients for BM prevention trials.