PurposeTo further improve analysis of the two-flash multifocal electroretinogram (2F-mfERG) in glaucoma in regard to structure–function analysis, using discrete wavelet transform (DWT) analysis.MethodsSixty subjects [35 controls and 25 primary open-angle glaucoma (POAG)] underwent 2F-mfERG. Responses were analyzed with the DWT. The DWT level that could best separate POAG from controls was compared to the root-mean-square (RMS) calculations previously used in the analysis of the 2F-mfERG. In a subgroup analysis, structure–function correlation was assessed between DWT, optical coherence tomography and automated perimetry (mf103 customized pattern) for the central 15°.ResultsFrequency level 4 of the wavelet variance analysis (144 Hz, WVA-144) was most sensitive (p < 0.003). It correlated positively with RMS but had a better AUC. Positive relations were found between visual field, WVA-144 and GCIPL thickness. The highest predictive factor for glaucoma diagnostic was seen in the GCIPL, but this improved further by adding the mean sensitivity and WVA-144.ConclusionsmfERG using WVA analysis improves glaucoma diagnosis, especially when combined with GCIPL and MS.