Abstract: Objective: Seven nucleosides, including adenine, adenosine, cytidine, hypoxanthine, guanosine, uracil and 2'- deoxyadenosine, were determined and analyzed by HPLC in the root bark of Morus australis, Morus cathayana and Morus alba from different areas, so as to provide reference for formulating quality standards. Methods: A venusil MP C₁₈ (5 μm, 250 mm × 4.6 mm) column was used. The mobile phases of methanol and water were used for gradient elution. Detection wavelength was 260 nm. The flow rate was 1.0 mL/min. The column temperature was 30 ℃. Results: There was a good linear relationship between the seven nucleosides in their respective concentration ranges (r≥0.9998). The average recoveries were 97.49%~101.45% with RSD ≤3%. There were significant differences in the contents of seven nucleosides in the root bark of Morus australis, Morus cathayana and Morus alba from different producing areas. The contents of uracil and hypoxanthine in the root bark of Morus alba and Morus australis were higher, but the contents of hypoxanthine in different varieties were different, and the content of cytidine was the lowest. The content of guanosine and adenine in root bark of Morus alba was higher than that of 2'- deoxyadenosine. The nucleoside composition of Morus australis and Morus cathayana was slightly lower than that of Morus alba. The results of independent sample t-test showed that there was no significant difference among the seven nucleosides in the root bark of Morus australis, Morus cathayana and Morus alba (P>0.05) and its nucleoside components had certain isotropy. The results of principal component analysis and cluster analysis showed that Morus australis, Morus cathayana and Morus alba could not be distinguished according to the difference of nucleoside content, indicating that Australis Cortex Mori from Morus australis, Morus cathayana could be used as the substitute of Cortex Mori. Conclusion: There is no significant difference in nucleoside contents among the root bark of Morus australis, Morus cathayana and Morus alba. This study provides a scientific basis for the study on the quality equivalence of Morus australis, Morus cathayana and Morus alba.