Background: Recent studies indicate that increased expression of glyoxalase I (GLO1) could result in epileptic seizures; thus, this study further explored the association of GLO1 with epilepsy from the perspective of molecular genetics. Material and methods: GLO1 single nucleotide polymorphisms (SNPs; rs1130534, rs4746 and rs1049346) were investigated in cohort I (the initial samples: 249 cases and 289 controls). A replication study designed to confirm the positive findings in cohort I was performed in cohorts II (the additional samples: 130 cases and 191 controls) and I + II. Results: In cohorts I, II and I + II, the CC genotype at rs1049346 T > C exerts a protective effect against both late onset epilepsy (odds ratio [OR] = 2.437, p = 0.013; OR = 2.844, p = 0.008; OR = 2.645, p = 0.000, q = 0.003, respectively) and drug-resistant epilepsy (DRE) (OR = 2.985, p = 0.020; OR = 2.943, p = 0.014; OR = 3.049, p = 0.001, q = 0.006, respectively). Further analyses in cohort I + II indicate that the presence of the TAC/AAT haplotypes (rs1130534-rs4746-rs1049346) may be used as a marker of predisposition to/protection against DRE (p = 0.002, q = 0.010; p = 0.000, q = 0.002, respectively). Conclusions: This study is the first to demonstrate that the GLO1 SNPs are significantly associated with epilepsy. In particular, the rs1049346 T > C SNPs are potentially useful for risk assessment of late-onset epilepsy and DRE. (C) 2016 Elsevier B.V. All rights reserved.