Abstract: N⁶-methyladenine (6mA) is a type of DNA methylation epigenetic modification. In recent years, the development of third generation sequencing technology has led to many important advances in 6mA research at the whole genome level. However, a few of comparative m6A methylome studies have been performed in close related species. In order to reveal the distribution of 6mA and the changes in close related species, we isolated and sequenced the macronuclei genome of Tetrahymena malaccensis and Tetrahymena pyriformis using PacBio SMRT sequencing techonology. Combining with the previous published Tetrahymena thermophila sequencing data, comparative 6mA methylome analysis in the three species of Tetrahymena was carried out, and we found that: (1) The 6mA methylation site distribution characteristics in the three species of Tetrahymena were similar, including periodic distribution about 200 bp and conservative AT motif; (2) 6mA was mainly distributed at the 5' of the gene body, and the 6mA distributed in approximate regions in gene body among orthologs, but showed low conservation at the single nucleotide level; (3) In recent duplicated inparalogs, the 6mA distribution showed high single nucleotide level conservation; (4) According to the divergence time of the three species of Tetrahymena, the speed of new 6mA sites establishment was about 69.9—226 sites per Mb sequences per million years.