The adaptive optics scanning laser ophthalmoscope has been fitted with three light sources of different wavelengths to allow simultaneous or separate imaging with one, two or three wavelength combinations. The source wavelengths used are 532 nm, 658 nm and 840 nm. Typically the instrument is used in dual-frame mode, performing imaging at 840 nm and precisely coincident retinal stimulation in one of the visible wavelengths. Instrument set-up and single-detector image capture are described. Simultaneous multi-wavelength imaging in the living human retina is demonstrated. The chromatic aberrations of the human eye lead to lateral and axial shifts, as well as magnification differences in the image, from one wavelength to another. Measurement of these chromatic effects is described for instrument characterization purposes.