The retinal pigment epithelium (RPE) is vital for the healthy function of the retina. Cellular level changes in the RPE are not visualized with current clinical techniques due to a lack of spatial resolution. Fluorescence adaptive optics scanning light ophthalmoscopy (AOSLO) can image RPE cells by utilizing their intrinsic autofluorescence (AF). The RPE AF has been imaged with only a few discrete excitation and emission bands and the multi-spectral AF has not been interrogated systematically at the level of single cells. In this study, we imaged 16 healthy eyes (ages 20-75) with AOSLO to investigate the multi-spectral AF as a function of age and wavelength with excitation from 650 - 805 nm. Quantitative analysis showed that 720 nm light produced images with the highest SNR (65.0 dB). Spatial AF variability showed a trend to increase with aging, suggesting increased heterogeneity in RPE AF with age. Spatial variability in the multi-spectral fluorescence of RPE cells with age may be a consequence of normal age-related loss of RPE cells. Multi-spectral fluorescence AOSLO provides new insight into aging related changes to RPE cells and may be a useful tool for studying diseases that affect the RPE, such as age-related macular degeneration (AMD).