To develop a non-invasive method for quantification of blood and pigment distributions across the posterior pole of the fundus from multispectral images using a computer-generated reflectance model of the fundus.A computer model was developed to simulate light interaction with the fundus at different wavelengths. The distribution of macular pigment (MP) and retinal haemoglobins in the fundus was obtained by comparing the model predictions with multispectral image data at each pixel. Fundus images were acquired from 16 healthy subjects from various ethnic backgrounds and parametric maps showing the distribution of MP and of retinal haemoglobins throughout the posterior pole were computed.The relative distributions of MP and retinal haemoglobins in the subjects were successfully derived from multispectral images acquired at wavelengths 507, 525, 552, 585, 596, and 611 nm, providing certain conditions were met and eye movement between exposures was minimal. Recovery of other fundus pigments was not feasible and further development of the imaging technique and refinement of the software are necessary to understand the full potential of multispectral retinal image analysis.The distributions of MP and retinal haemoglobins obtained in this preliminary investigation are in good agreement with published data on normal subjects. The ongoing development of the imaging system should allow for absolute parameter values to be computed. A further study will investigate subjects with known pathologies to determine the effectiveness of the method as a screening and diagnostic tool.