Abstract: ObjectiveDifferent strains of Exserohilum rostratum,causing exserohilum banana leaf spot disease were characterized at molecular level using the conserved sequence of Brn1 gene.Method Brn1 gene of three pathogenic isolates (CLER09,D087 and JL05) of E.rostratum causing exserohilum banana leaf spot disease was amplified using primer pair Brn1 01 (5'-GCCAACATCGCAAACATGG-3') and Brn1 02 (5'-GCAAGCAGCACCGTCAATACCAAT-3'),and phylogenetic analysis was carried out.Result The results showed absence and substitute of single bases in Brn1 sequences among different isolates of E.rostratum isolated from diseased banana plant and sequence collected from GenBank database.The isolates collected from banana were clustered with E.rostratum in an absolute evolution group in the phylogenetic tree,and significantly distinguished from other species of Exserohilum spp.Analysis result of the genetic distance matrix showed that tested isolates were genetically close to E.rostratum sequences collected from GenBank database with intraspecies genetic distance of 0.000-0.018 and interspecies genetic distance of 0.020-0.093 in Exserohilum genus. Conclusion The obvious genetic difference among isolates of E.rostratum could be considered as intraspecific variation and the Brn1 gene sequence may be used for molecular evidence for supporting the isolates with markedly morphological difference as members of E.rostratum.