To identify whether there are functional abnormalities in the retina of amblyopic eyes using multifocal electroretinography (mfERG).This is a prospective study of patients ≥ 7 years of age identified with unilateral amblyopia (strabismic or anisometropic). Multifocal ERG and flash ERG were performed to compare parameters between the amblyopic and non-amblyopic fellow eyes. A complete analysis of the five ring averages was done including the central ring.Thirty-eight patients were included: mean age was 14.3 ± 7.3 years; 18 patients were strabismic and 20 were anisometropic. Amblyopic eye responses across the rings in multifocal ERG were diminished compared with fellow non-amblyopic eyes with significant differences detected in the central rings (p = 0.001). On the other hand, flash ERG did not show any consistently significant differences. When divided by severity, amplitudes of central rings were significantly lower in severely amblyopic eyes compared with non-amblyopic eyes (p = 0.001), while in mild amblyopia, no significant differences were observed. No significant difference was observed between anisometropic and strabismic amblyopic eyes.Using multifocal ERG, significantly decreased amplitudes were observed in amblyopic eyes compared with normal fellow eyes in the central ring. This correlated with the severity of amblyopia. No difference was observed when comparing the two groups of amblyopia (strabismic and anisometropic). Those findings may help clarify the pathophysiology of amblyopia better and open the door for new objective ways to monitor the response to amblyopia treatment but this needs to be further studied.