Taxonomic classification of whole eukaryotes using conserved proteins or nucleotide sequences that are used as criteria looks convenient and easy. However, these methods may not be reliable to long evolutionary history if their function is developed or degenerated in their life history. The sequence-based classification methods depending on the functional conservation may cause unknown bias. In this study, we propose novel classification using intron positions in a gene, which are not related to function. Intron positions of highly conserved ribosomal protein RPS13 gene were selected as novel criteria for the classification of whole eukaryotes from lower unicellular amoeba, algae, and flagellates to plants and animals. RPS13 gene encodes 151 amino acids and possesses 453 bp exon sequence in many eukaryotic organisms. The intron positions are determined based on their coding exon sequences, in which all the introns are designated by the positions from the first nucleotide of the start codon and the position numbers are attached with ‘i’ for intron indication. For example, human RPS13 gene contains introns at the positions of 24i, 73i, 152i, 322i, and 423i sites on the coding sequence. Interestingly, all the Deuterostomia animals including starfish, leech, and octopus showed the same intron positions to human. Similarly, all the land plants showed 24i, 97i, 236i, and 423i positions. In lower unicellular eukaryotes, which showed nonintron or variety of positions, only amoeba Lenisia limosa has the intron position of 152i site and Chlorophyta Pedinophyceae sp. has single 97i position, suggesting that they may be the ancestral organisms of animals and plants, respectively. As a conclusion, intron positions of RPS13 gene can be used as a guide for the taxonomic classification of eukaryotic organisms