Genetic relationships among 50 Gentiana accessions, comprising 36 wild species and 14 cultivars, were determined based on analysis of sequence data for the chloroplast trnL(UAA) intron, the rpl16 coding region and the rpl16-rpl14 intergenic spacer (IGS), together with nuclear DNA content as determined by flow cytometric analysis. The combined chloroplast DNA (cpDNA) data set was analyzed using both neighbor-joining (NJ) and maximum parsimony (MP) methods. The NJ and the strict consensus trees were generally congruent with previous phylogenetic and taxonomic studies, whereas G. cachemirica and G. yakushimensis were classified in different sectional affinities from their prevailing classifications. Three major cpDNA haplotypes (designated A, B and C), comprising 30 accessions in the sections Pneumonanthe, Cruciata, and Kudoa (ser. Monanthae), were each distinguished by two single-nucleotide polymorphisms in the rpl16-rpl14 IGS and the rpl16 coding region. Nuclear DNA content varied from 6.47 to 11.75 pg among the taxa possessing cpDNA haplotype A. These results provide genetic information that will assist in the development of future Gentiana breeding strategies.