Objective To combine two-wavelength fundus autofluorescence (FAF) with multifocal electroretinography (mf-ERG) in retinitis pigmentosa (RP) and discuss the microstructural abnormalities as seen with spectral-domain optical conherence tomography (Spectralis OCT). Methods This was a noninterventional, observational study, based on the observation and analysis of a series of cases. Fifteen eyes of 8 patients with RP were evaluated with Spectralis OCT, blue light fundus autofluorescence (BL-FAF, excitation 488 nm, emission ＞500 nm) and near-infrared fundus autofluorescence (NIR-FAF, excitation 787 nm, emission ＞800 nm). BL-FAF and NIR-FAF imaging were performed with a confocal scanning laser ophthalmoscope (Heidelberg Retina Angiograph 2). Five of 8 patients received fundus fluorescein angiography (FFA) while 3 also had indocyanine green angiography (ICGA) simultaneously. Seven had fundus photography and mf-ERG was performed on 4 cases. The FAF distribution in RP was observed and the correlated changes of microstructural abnormalities were analyzed with Spectralis OCT and the response (Amp.P1) in mf-ERG. Results BL-FAF and NIR-FAF showed a hypoautofluorescent pattern in the atrophied area of the RPE and photoreceptor and bone spicule pigmentation, where mf-ERG showed that the peak in the fovea had disappeared and responses had markedly decreased throughout the macula. Spectralis OCT demonstrated that normal FAF demarcated the border within an area of preserved PR and RPE, while outer limiting membrane (OLM) showed disturbances. In several cases, the hyperautofluorescent pattern of NIR-FAF was less obvious than that of BL-FAF in these areas. In FFA images showing early stages, a window defect was seen at the posterior pole, suggesting RPE malfunction and in ICGA images of early stages, choroidal capillary atrophy was observed. Conclusion The two-wavelength FAF, Spectralis OCT and mf-ERG are useful noninvasive diagnostic tools to identify retinitis pigmentosa. The change in these measures indicate a common pathway for photoreceptor and RPE degeneration involving lipofuscin and non-lipofuscin fluorophores such as melanin (oxidation) with different pathophysiologic processes by BL-FAF and NIR-FAF.