Background: Anisometropia is associated with asymmetric ocular growth and may influence retinal structure, yet its impact on retinal nerve fiber layer thickness (RNFLT) remains incompletely understood. This study aimed to evaluate RNFLT and central macular thickness (CMT) in children with anisometropia and to examine their relationships with refractive error and ocular biometric parameters. Methods: This cross-sectional study included children aged 5–16 years with anisometropia. Comprehensive ophthalmic evaluation included cycloplegic refraction, ocular biometry, and spectral-domain optical coherence tomography. Peripapillary RNFLT and CMT were measured and compared between worse and fellow eyes, as well as between amblyopic and non-amblyopic eyes. Correlations between spherical equivalent refraction (SER) and ocular parameters, including axial length, intraocular pressure, CMT, and RNFLT, were assessed. Results: Among 46 children (median age 14 years), 45.7% (n = 21) had anisometropic amblyopia. Nasal RNFLT was significantly greater in worse eyes compared with fellow eyes (P < 0.05), while other quadrants and CMT showed no significant interocular differences. Amblyopic eyes showed higher RNFLT values than non-amblyopic eyes, reaching significance only in the nasal quadrant. SER showed a strong negative correlation with axial length (r = -0.91, P < 0.001) and moderate or weak positive correlations with quadrant-specific RNFLT, including inferior (r = +0.56, P = 0.001), superior (r = +0.67, P < 0.001), temporal (r = +0.59, P < 0.001), and nasal (r = +0.37, P = 0.012) quadrants, but not with CMT (r = -0.22, P > 0.05) or IOP (r = +0.19, P > 0.05). Conclusions: Children with anisometropia exhibit selective regional RNFLT alterations, particularly involving the nasal quadrant, while macular thickness remains largely preserved. The observed associations between refractive error, AL, and RNFLT suggest that anisometropia may influence retinal structural development in a region-specific manner. Longitudinal studies are warranted to clarify the temporal relationship between refractive asymmetry and retinal structural remodeling.