Sugarcane bacilliform virus (SCBV) is an economically important virus limiting sugarcane production worldwide. Although Ethiopia is a major sugarcane producer, and virus-like symptoms are frequently observed in sugarcane fields, there is a complete lack of information as to the occurrence, distribution and molecular properties of SCBV. This study was aimed to identify and characterize SCBV isolates in Ethiopia using molecular methods. Out of 292 leaf samples collected and tested by PCR, 76 samples (26% incidence level) were found SCBV-positive. Nucleotide sequence analysis results showed that three Ethiopian isolates (SCBV-EtS3, SCBV-EtS6 and SCBV-EtC10) shared high level of nucleotide identity (99.5-100%) among themselves and with SCBV isolates from China (accession numbers MH037614 and MH037915). Another isolate, SCBV-EtC2, shared maximum identity of 78% with the other three SCBV isolates from Ethiopia and 99.8% with SCBV isolates from China (KM214357 and KM214307). Based on phylogenetic analysis, isolates from Ethiopia were segregated into two different clusters. Isolates SCBV-EtS3, SCBV-EtS6 and SCBV-EtC10 clustered with SCBV-Q group and SCBV-EtC2 with SCBV-H group. This study provides information on the occurrence of SCBV for the first time in Ethiopia and also contributes to the understanding of the genetic diversity of SCBV. Keywords: Caulimoviridae; RNase H, Saccharum spp.; Sugarcane bacilliform virus.