Posttranscriptional methylation of RNA cytosine residues to 5-methylcytosine (m⁵C) is an important modification with diverse roles, such as regulating stress responses, stem cell proliferation, and RNA metabolism. Here, we used RNA bisulfite sequencing for transcriptome-wide quantitative mapping of m⁵C in the model plant Arabidopsis thaliana. We discovered more than a thousand m⁵C sites in Arabidopsis mRNAs, long noncoding RNAs, and other noncoding RNAs across three tissue types (siliques, seedling shoots, and roots) and validated a number of these sites. Quantitative differences in methylated sites between these three tissues suggest tissue-specific regulation of m⁵C. Perturbing the RNA m⁵C methyltransferase TRM4B resulted in the loss of m⁵C sites on mRNAs and noncoding RNAs and reduced the stability of tRNA^Asp(GTC). We also demonstrate the importance of m⁵C in plant development, as trm4b mutants have shorter primary roots than the wild type due to reduced cell division in the root apical meristem. In addition, trm4b mutants show increased sensitivity to oxidative stress. Finally, we provide insights into the targeting mechanism of TRM4B by demonstrating that a 50-nucleotide sequence flanking m⁵C C3349 in MAIGO5 mRNA is sufficient to confer methylation of a transgene reporter in Nicotiana benthamiana.