RNA has co-evolved with numerous post-transcriptional modifications to sculpt interactions with proteins and other molecules in animals and plants. Methylation is the most abundant post-transcriptional RNA modification occurring both on adenine (m6A) and cytosine (m5C). Here we phenotypically and molecularly characterize NSUN2 and NOP2 that are two RNA methyltransferases that catalyzes RNA 5-methylcytosine (m5C) in plants. We discovered Arabidopsis thaliana nsun2b mutant seedlings are more sensitive to oxidative stress and have shorter roots than wild type as a consequence of reduced cell division. To identify RNA m5C sites catalysed by NSUN2B, we performed bisulfite conversion and transcriptome wide Illumina sequencing (BS-RNA-seq) on wild type and nsun2b seedlings. We discovered >1,000 potential m5C sites in mRNAs, long non-coding RNAs, rRNAs and tRNAs with at least 2% methylation in wild type seedlings. Most of these m5C sites in mRNAs, lncRNAs and tRNAs were absent in nsun2b. Thirty-four methylated nuclear tRNAs were detected, containing 39 potential m5C sites, which occur at six structural positions. Of these 39 potential sites, 3 required the RNA methyltransferase TRDMT1 (tRNA aspartic acid methyltransferase 1), and 32 absolutely required or had reduced methylation in nsun2b mutants. Interestingly, no m5C sites were detected in mitochondrial or chloroplast encoded tRNAs. NOP2 has three paralogues, NOP2A, NOP2B and NOP2C and here we show nop2a/nop2b mutants are embryo lethal demonstrating an essential biological function. On going work is determining the sites methylated by NOP2A and NOP2B. Our results demonstrate RNA m5C is a genome-wide modification and is an essential post-transcriptional modification in plants.