The F0 and FM level fluorescence from a wild-type barley, a Chl b-less mutant barley, and a maize leaf was determined from 430 to 685 nm at 10 nm intervals using pulse amplitude-modulated (PAM) fluorimetry. Variable wavelengths of the pulsed excitation light were achieved by passing the broadband emission of a Xe flash lamp through a birefringent tunable optical filter. For the three leaf types, spectra of FV/FM (=(FM − F0)/FM) have been derived: within each of the three spectra of FV/FM, statistically meaningful variations were detected. Also, at distinct wavelength regions, the FV/FM differed significantly between leaf types. From spectra of FV/FM, excitation spectra of PS I and PS II fluorescence were calculated using a model that considers PS I fluorescence to be constant but variable PS II fluorescence. The photosystem spectra suggest that LHC II absorption results in high values of FV/FM between 470 and 490 nm in the two wild-type leaves but the absence of LHC II in the Chl b-less mutant barley leaf decreases the FV/FM at these wavelengths. All three leaves exhibited low values of FV/FM around 520 nm which was tentatively ascribed to light absorption by PS I-associated carotenoids. In the 550–650 nm region, the FV/FM in the maize leaf was lower than in the barley wild-type leaf which is explained with higher light absorption by PS I in maize, which is a NADP-ME C4 species, than in barley, a C3 species. Finally, low values of FV/FM at 685 in maize leaf and in the Chl b-less mutant barley leaf are in agreement with preferential PS I absorption at this wavelength. The potential use of spectra of the FV/FM ratio to derive information on spectral absorption properties of PS I and PS II is discussed.