The Iris series Chinenses in Korea comprises four species (I. minutoaurea, I. odaesanensis, I. koreana, and I. rossii), and the group includes some endangered species, owing to their high ornamental, economic, and conservation values. Among them, the putative allotetraploid, Iris koreana (2n = 4x = 50), is hypothesized to have originated from the hybridization of the diploids I. minutoaurea (2n = 2x = 22) and I. odaesanensis (2n = 2x = 28) based on morphological characters, chromosome numbers, and genome size additivity. Despite extensive morphological and molecular phylogenetical studies on the genus Iris, little is known about Korean irises in terms of their complete chloroplast (cp) genomes and molecular cytogenetics that involve rDNA loci evolution based on fluorescence in situ hybridization (FISH). This study reports comparative analyses of the karyotypes of the three Iris species (I. koreana, I. odaesanensis, and I. minutoaurea), with an emphasis on the 5S and 35S rDNA loci number and localization using FISH together with the genome size and chromosome number. Moreover, the cp genomes of the same individuals were sequenced and assembled for comparative analysis. The rDNA loci numbers, which were localized consistently at the same position in all species, and the chromosome numbers and genome size values of tetraploid Iris koreana (four 5S and 35S loci; 2n = 50; 1C = 7.35 pg) were additively compared to its putative diploid progenitors, I. minutoaurea (two 5S and 35S loci; 2n = 22; 1C = 3.71 pg) and I. odaesanensis (two 5S and 35S loci; 2n = 28; 1C = 3.68 pg). The chloroplast genomes were 152,259-155,145 bp in length, and exhibited a conserved quadripartite structure. The Iris cp genomes were highly conserved and similar to other Iridaceae cp genomes. Nucleotide diversity analysis indicated that all three species had similar levels of genetic variation, but the cp genomes of I. koreana and I. minutoaurea were more similar to each other than to I. odaesanensis. Positive selection was inferred for psbK and ycf2 genes of the three Iris species. Phylogenetic analyses consistently recovered I. odaesanensis as a sister to a clade containing I. koreana and I. minutoaurea. Although the phylogenetic relationship, rDNA loci number, and localization, together with the genome size and chromosome number of the three species, allowed for the inference of I. minutoaurea as a putative maternal taxon and I. odaesanensis as a paternal taxon, further analyses involving species-specific molecular cytogenetic markers and genomic in situ hybridization are required to interpret the mechanisms involved in the origin of the chromosomal variation in Iris series Chinenses. This study contributes towards the genomic and chromosomal evolution of the genus Iris.