YTH domain-containinggenes are important readers ofN6-methyladenosine (m6A) modifications with ability to directly affect the fates of distinct RNAs in organisms. Despite their importance, little is known aboutYTH domain-containinggenes in teleosts until now. In the present study, a total of 10YTH domain-containinggenes have been systematically identified and functionally characterized in rainbow trout (Oncorhynchus mykiss). According to the phylogenetic tree, gene structure and syntenic analysis, theseYTH domain-containinggenes could be classified into three evolutionary subclades, including YTHDF, YTHDC1 and YTHDC2. Of them, the copy number ofOmDF1,OmDF2,OmDF3, andOmDC1were duplicated or even triplicated in rainbow trout due to the salmonid-specific whole-genome duplication event. The three-dimensional protein structure analysis revealed that there were similar structures and the same amino acid residues that were associated with cage formation between humans and rainbow trout, implying their similar manners in binding to m6A modification. Additionally, the results of qPCR experiment indicated that the expression patterns of a fewYTH domain-containinggenes, especiallyOmDF1b,OmDF3aandOmDF3b, were significantly different in liver tissue of rainbow trout under four different temperatures (7 °C, 11 °C, 15 °C, and 19 °C). The expression levels ofOmDF1a,OmDF1bandOmDC1awere obviously repressed in spleen tissue of rainbow trout at 24 h afterYersinia ruckeriinfection, while increased expression was detected inOmDF3b. This study provides a systemic overview ofYTH domain-containinggenes in rainbow trout and reveals their biological roles in responses to temperature stress and bacterial infection.