This research confirms and further establishes that pathogenic variants in a fourth gene, METTL23, are associated with autosomal dominant normal-tension glaucoma (NTG). To determine the frequency of glaucoma-causing pathogenic variants in the METTL23 gene in a cohort of patients with NTG from Iowa. This case-control study took place at a single tertiary care center in Iowa from January 1997 to January 2024, with analysis occurring between January 2023 and January 2024. Two groups of participants were enrolled from the University of Iowa clinics: 331 patients with NTG and 362 control individuals without glaucoma. Patients with a history of trauma; steroid use; stigmata of pigment dispersion syndrome; exfoliation syndrome; or pathogenic variants in MYOC, TBK1, or OPTN were also excluded. Detection of an enrichment of METTL23 pathogenic variants in individuals with NTG compared with control individuals without glaucoma. The study included 331 patients with NTG (mean [SD] age, 68.0 [11.7] years; 228 [68.9%] female and 103 [31.1%] male) and 362 control individuals without glaucoma (mean [SD] age, 64.5 [12.6] years; 207 [57.2%] female and 155 [42.8%] male). There were 5 detected instances of 4 unique METTL23 pathogenic variants in patients with NTG. Three METTL23 variants-p.Ala7Val, p.Pro22Arg, and p.Arg63Trp-were judged to be likely pathogenic and were detected in 3 patients (0.91%) with NTG. However, when all detected variants were evaluated with either mutation burden analysis or logistic regression, their frequency was not statistically higher in individuals with NTG than in control individuals without glaucoma (1.5% vs 2.5%; P = .27). This investigation provides evidence that pathogenic variants in METTL23 are associated with NTG. Within an NTG cohort at a tertiary care center, pathogenic variants were associated with approximately 1% of NTG cases, a frequency similar to that of other known normal-tension glaucoma genes, including optineurin (OPTN), TANK-binding kinase 1 (TBK1), and myocilin (MYOC). The findings suggest that METTL23 pathogenic variants are likely involved in a biologic pathway that is associated with glaucoma that occurs at lower intraocular pressures.