Citrus tristeza virus (CTV), the most devastating viral pathogen in citrus, causes tremendous economic losses to citrus industry worldwide. The CTV isolates exhibit variable pathogenicities on their hosts indicating a mixed population of the CTV in nature. Several fragments within the CTV genome have been used for studying the genetic diversity of the CTV, however, the best region for rapid the CTV strain differentiation is still absent at present. In present study, a systemic analysis was carried out to evaluate the best region within the CTV genome for rapid CTV strain differentiation. Results of our study showed that the major coat protein (CP) coding region was the best region for this purpose. Using pair-wise distance frequency distribution plot, a reasonable genetic distance cut-off value was set for the CTV CP gene for the CTV strain differentiation. Using this criterion, eight CTV strains, including seven well characterized and a new strain, were successfully differentiated using 537 CTV isolates reported from 38 countries. The global strain distribution pattern was then determined and discussed. Our results also provided a new insight into the evolution and spreading of the virus, as well as the information for developing proper disease management strategy. Citrus tristeza virus (CTV), the most devastating viral pathogen in citrus, causes tremendous economic losses to citrus industry worldwide. The CTV isolates exhibit variable pathogenicities on their hosts indicating a mixed population of the CTV in nature. Several fragments within the CTV genome have has used for studying the genetic diversity of the CTV, however, the best region for rapid the CTV strain differentiation is still absent at present. In present study, a systemic analysis was carried out to evaluate the best region within the CTV genome for rapid CTV strain differentiation. Results of our study showed that the major coat protein (CP) coding region was the best region for this purpose. Using pair-wise distance frequency distribution plot, a reasonable genetic distance cut-off value was set for the CTV CP gene for the CTV strain differentiation. Using this criterion, eight CTV strains, including seven well characterized and a new strain, were successfully differentiated usi ng 537 CTV isolates reported from 38 countries. The global strain distribution pattern was then determined and discussed. Our results also provided a new insight into the evolution and spreading of the virus, as well as the information for developing proper disease management strategy.