Spectral tuning of rhodopsins commonly refers to the effects of opsin amino acid substitutions on the wavelength for peak sensitivity of the rhodopsin absorption spectrum. Nymphalini butterflies provide an opportunity for identifying some of the amino acid substitutions responsible for insect rhodopsin spectral tuning because the majority of photoreceptor cells (R3-9) in the adult retina express only a single long wavelength-sensitive (LWS) opsin mRNA transcript. Therefore, the opsin genotype can be directly correlated with its phenotype. We determined the LWS opsin gene sequence from cDNA of the mourning cloak Nymphalis antiopa, and from genomic DNA of the malachite Siproeta stelenes and the peacock Inachis io. Using an epi-microspectrophotometer we examined each butterfly's eyeshine for photochemical evidence of multiple LWS rhodopsins and found only one. We then performed partial-bleaching experiments to obtain absorbance spectra for the LWS rhodopsins of all three species as well as from another nymphalid, the buckeye Junonia coenia. The isolated LWS opsin gene sequences varied in length from 1437-1612 bp and encode rhodopsins R522 (S. stelenes), R530 (I. io), R534 (N. antiopa) and, together with a previously published sequence, R510 (J. coenia). Comparative sequence analysis indicates that the S. stelenes rhodopsin is slightly blue-shifted compared to the typical 530 nm lepidopteran rhodopsin because of the presence of a S138A substitution at a homologous site that in mammalian MWS/LWS rhodopsins causes a 5 nm blue-shift. The difference in peak absorption between R522 of S. stelenes and R530 of Inachis io is therefore largely accounted for by this substitution. This suggests that spectral tuning mechanisms employing the S138A may have evolved in parallel in mammalian and butterfly MWS/LWS rhodopsins across 500 million years of evolution.