Non-small cell lung cancer (NSCLC) often metastasizes to the brain, but identifying which patients will develop brain metastases (BM) is difficult. 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 seven autophagy-related genes (ATG3, ATG5, ATG7, ATG10, ATG12, ATG16L1, and 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.