Studies of RNA modification are usually focused on tRNA. However the modification of other small RNAs, including 5.8S rRNA, 5S rRNA, and small RNA sized at 10-60 nt, is still largely unknown. In this study, we established an efficient method based on liquid chromatography-tandem mass spectrometry (LC-MS/MS) to simultaneously identify and quantify more than 40 different types of nucleosides in small RNAs. With this method, we revealed 23 modified nucleosides of tRNA from mouse liver, and 6 of them were observed for the first time in eukaryotic tRNA. Moreover, 5 and 4 modified nucleosides were detected for the first time in eukaryotic 5.8S and 5S rRNA, respectively, and 22 modified nucleosides were identified in the small RNAs sized at 30-60 or 10-30 nt. Interestingly, two groups of 5S rRNA peaks were observed when analyzed by HPLC, and the abundance of modified nucleosides is significantly different between the two groups of peaks. Further studies show that multiple modifications in small RNA from diabetic mouse liver are significantly increased or decreased. Taken together, our data revealed more modified nucleosides in various small RNAs and showed the correlation of small RNA modifications with diabetes. These results provide new insights to the role of modifications of small RNAs in their stability, biological functions, and correlation with diseases.