Studies investigating the spectral reflectance of coral reef benthos and substrates have focused on the measurement of pure endmembers, where the entire field of view (FOV) of a spectrometer is focused on a single benthos or substrate type. At the spatial scales of the current satellite sensors, the heterogeneity of coral reefs even at a sub-metre scale means that many individual image pixels will be made up of a mixture of benthos and substrate types. If pure endmember spectra are used as training data for image classification, there is a spatial discrepancy, because many pixels will have a mixed endmember spectral reflectance signature. This study investigated the spectral reflectance of coral reef benthos and substrates at a spatial scale directly linked to the pixel size of high spatial resolution imaging systems, by incorporating multiple benthos and substrate types into the spectrometer FOV in situ. A total of 334 spectral reflectance signatures were measured of 19 assemblages of the coral reef benthos and substrate types. The spectra were analysed for separability using first derivative values, and a discrimination decision tree was designed to identify the assemblages. Using the decision tree, it was possible to identify 15 assemblages with a mean overall classification accuracy of 62.6%.