The C5-methylation of uracil to form 5-methyluracil (m⁵U) is a ubiquitous base modification of nucleic acids. Four enzyme families have converged to catalyze this methylation using different chemical solutions. Here, we investigate the evolution of 5-methyluracil synthase families in Mollicutes, a class of bacteria that has undergone extensive genome erosion. Many mollicutes have lost some of the m⁵U methyltransferases present in their common ancestor. Cases of duplication and subsequent shift of function are also described. For example, most members of the Spiroplasma subgroup, use the ancestral tetrahydrofolate-dependent TrmFO enzyme, to catalyze the formation of m⁵U54 in tRNA, while a TrmFO paralog (termed RlmFO) is responsible for m⁵U1939 formation in 23S RNA. RlmFO has replaced the S-adenosyl-l-methionine (SAM)-enzyme RlmD that adds the same modification in the ancestor and which is still present in mollicutes from the Hominis subgroup. Another paralog of this family, the TrmFO-like protein, has a yet unidentified function that differs from the TrmFO and RlmFO homologs. Despite having evolved towards minimal genomes, the mollicutes possess a repertoire of m⁵U modifying enzymes that is highly dynamic and has undergone horizontal transfer. This emphasizes the necessity for combining bioinformatics predictions with empirical testing and structural information to get a reliable functional annotation of these enzymes.