Occurrence and distribution of citrus canker, caused by Xanthomonas axonopodis pv. citri, in the Rift Valley of Ethiopia had been reported. Studies were conducted with the objective of monitoring epidemiology of the disease in the country, identifying, characterizing and determining the genetic diversity of 80 Ethiopian strains on the basis of their pathogenicity test and molecular properties. Diagnosis was carried out using PCR. Pathogenecity tests, insertion sequence ligation mediated polymerase chain reaction (IS-LM- PCR) and multilocus variable number of tandom repeat analysis (MLVA) were carried out following conventional procedures established for same. The disease was observed to spread to central and northern part of the country. The expected 492-bp amplification products were produced with DNAs of all Ethiopian strains. Pathogenecity tests indicated canker symptoms on Mexican lime (Citrus aurantifolia) and alemow (Citrus macrophylla) leaves and not on sweet orange (Citrus sinensis) and grapefruit (Citrus paradis!). AII four primers in the IS-LM-PCR did not indicate satisfactory polymorphism within the strains. On the other hand, MLVA analysis indicated polymorphism between populations of the Ethiopian strains and those from international collections. On the basis of results from pathogenecity tests, ISLM-PCR, and MLVA analysis, the Ethiopian strains were grouped into a cluster only comprised of X.citri pv.citri pathotype A*, and is epidemiologically important on Mexican lime and alemow.