Purpose To determine the ability of blue-on-yellow multifocal visual evoked potentials (BonY mfVEP) to identify functional loss in preperimetric glaucoma. Design Prospective case series. Participants Thirty patients with glaucomatous optic discs and normal standard visual fields. Methods All patients underwent BonY mfVEP, dilated optic disc stereophotography, and optical coherence tomography (Fast RNFL protocol). Optic disc photographs were assessed by 2 independent examiners in a masked fashion. Main Outcome Measures The mfVEP amplitude asymmetry and latency values were analyzed and compared topographically with findings of disc assessment. Average retinal nerve fiber layer (RNFL) thickness, RNFL asymmetry, and sectors with RNFL thinning were compared between patients with and without mfVEP defects. Results Fourteen (46.7%) patients demonstrated significant abnormality on amplitude asymmetry deviation plots of BonY mfVEP. In all 14 cases, the defect was monocular and corresponded to the eye with the worse disc. In 13 of 14 patients, the defect also corresponded to the location of the worst affected rim. Average RNFL thickness of eyes with mfVEP defects was 81.2±9.9 μm, significantly lower than that of patients without defects (90±10.5 μm; P = 0.035). Mean asymmetry of RNFL (better minus worse eye) also was significantly higher for patients with mfVEP defects compared with those without such defects (9.0±6.4 μm vs. 3.0±7 μm; P = 0.03). Average latency of both eyes of glaucomatous patients was delayed compared with that of controls, with no difference in latency between worse and better eyes of glaucoma patients. There was no association of latency delay with either the location of disc changes or mfVEP amplitude defects. Conclusions Amplitude asymmetry of the BonY mfVEP seems to be a promising tool to identify functional loss in preperimetric glaucoma.