ABSTRACTMany RNA modifications have been detected in rRNA, tRNA and small noncoding RNA (sncRNA) as well as in low-abundance RNA species such mRNA. Although RNA modifications play roles in many cellular and biological processes in various domains of life, knowledge about the diversity and role of RNA modifications inToxoplasma gondiiis limited. In this study, RNA modifications in threeT. gondiistrains (RH type I, PRU type II, and VEG type III) with distinct virulence abilities were determined by liquid chromatography-tandem mass spectrometry. We compared the levels of modifications of four nucleotides in tRNA and sncRNA, characterized RNA modification patterns of differentT. gondiistrains, and determined the diversity of RNA modifications. We detected and quantified 22 modified nucleosides in both tRNA and sncRNA. Significant differences in the diversity of the modified nucleosides were found between the threeT. gondiistrains. RNA modifications were correlated with the expression of manyT. gondiivirulence proteins. Some of the identified modifications (e.g., 2’-O-methylinosine, pseudouridine) play a role in mediating the host-parasite interaction. These results provide novel insight into the global modifications in tRNA and sncRNA, and the diversity of RNA modifications betweenT. gondiistrains with different virulence backgrounds.IMPORTANCEAlthough RNA modifications play roles in many cellular and developmental processes in various domains of life, knowledge about the patterns and functions of RNA modifications inT. gondiiis limited. Here, a quantitative liquid chromatography-tandem mass spectrometry (LC-MS/MS) approach was used to study global RNA modifications inT. gondiistrains of distinct virulence backgrounds. We quantified 22 modified nucleosides in both tRNA and sncRNA. SignificantT. gondiistrain-specific differences in RNA modifications were detected. More tRNA modifications correlated withT. gondiivirulence proteins than sncRNA modifications. RNA modifications were significantly correlated with virulence proteins. Our data provide the first comprehensive profiling of the modifications tRNA and sncRNA inT. gondii, expanding the diversity of RNA modifications in this parasite and suggesting new regulators for modulating its virulence.