AIM: To investigate the differences in retinal refraction difference values (RDVs) of adult patients with myopic anisometropia compared with those without myopic anisometropia, and to investigate the relationship between ocular biometric measurements and relative peripheral refraction. METHODS: This clinical observation study included 130 patients with myopia (-0.25 to -10.00 D) between October 2022 and January 2023 aged between 18 and 40y. The patients were divided into anisometropia (n=63; difference in binocular anisometropia ≥1.00 D) and non-anisometropia (n=67; difference in binocular anisometropia <1.00 D) groups accordingly. Ocular biometric measurements were performed by optical biometrics and corneal topography to assess the steep keratometry (Ks), flap keratometry (Kf), axial length (AL), corneal astigmatism (CYL; Ks-Kf), surface regularity index (SRI), surface asymmetry index (SAI), and central corneal thickness (CCT). The RDV was measured at five retinal areas from the fovea to 53 degrees (RDV-0-10, RDV-10-20, RDV-20-30, RDV-30-40, and RDV-40-53), the total RDV (TRDV) of 53 degrees, and four regions, including RDV-superior, RDV-inferior, RDV-temporal, and RDV-nasal. An analysis of Spearman correlation was carried out to examine the correlation between RDV and the spherical equivalent (SE) and ocular biological parameters. RESULTS: Within RDV-20-53, both groups showed relative hyperopic defocus, and the increase in RDV corresponds to the increase in eccentricity. In the myopic anisometropia group, the TRDV, RDV-20-53, RDV-superior, and more myopic eyes had significantly higher RDV-temporal values than less myopic eyes. (P<0.05). In the non-anisometropia group, there was no significant difference in the RDV between the more and less myopic eyes at different eccentricities (P>0.05). There was a negative correlation between SE and TRDV (r=-0.205, P=0.001), RDV-20-53 (r=-0.281, -0.183, -0.176, P<0.05), RDV-superior (r=-0.251, P<0.001), and RDV-temporal (r=-0.230, P<0.001), a negative correlation between CYL and RDV-10-30 (r=-0.147, -0.180, P<0.05), and a negative correlation between SRI and RDV-0-20 (r=-0.190, -0.170, P<0.05). AL had a positive correlation with RDV-20-30 (r=0.164, P=0.008) and RDV-temporal (r=0.160, P=0.010). CONCLUSION: More myopic eyes in patients with myopic anisometropia show more peripheral hyperopic defocus. Diopter and corneal morphology may affect peripheral retinal defocus.