Abstract Fraction I proteins were purified from 30 species of the genus Nicotiana, and separated into large and small subunits which in all cases displayed the same relative ratio. Tryptic peptides of each subunit which were soluble at pH 2.2 were analyzed by ion-exchange column chromatography with a gradient elution system. N. tabacum was used as a standard for comparison, 27 peptides being resolved from the large subunit and 23 for the small subunit. The composition of Fraction I proteins from other Nicotianas ranged from no differences (7 species) to as many 13 differences distributed in both the large and small subunits. The distribution of differences allowed a chemical taxonomic classification of the genus to be made which was closely similar (with only two excpetions) to the classical taxonomy. The results confirm a previous concept that mutation of the small subunit coded by nuclear DNA is much more frequent than mutation of the large subunit coded by chloroplast DNA. The differences also serve as unique phenotypic markers for both nuclear and chloroplast DNA genes coding for Fraction I protein. The markers will be of great utility in further studies of evolution, transfer of genetic information by parasexual hybridization and mode of chloroplast DNA mutation.