Contamination is a main issue in sugarcane tissue culture.The purpose of this study was to determine the contaminative bacteria frequently occurred during the production of sugarcane tissue culture.The gradient dilution method was used to isolate bacteria,and the toxonomic position was identified combining the biochemical,sequence alignment of 16S rDNA and phaC genes,phylogeomic analysis.The results showed that two type contaminative bacteria were obtained from the sugarcane tissue culture nutrient solution,and nine tested biochemical characters were the same between the two bacteria except mannitol utilization.Sequence alignment showed that intraspecific sequences of 16S rDNA and phaC genes were completely uniformity respectively in the two bacteria,but the difference of the two nucleotide bases in 16S rDNA and 23 in phaC sequences existed between the two bacteria.The phylogenic tree constructed based on 16S rDNA and phaC genes sequences respectively also indicated that the two bacteria were closest each other and clustered into two absolutely independent branches.Combining the biochemical characters,sequence alignment and phylogenetic analysis,the two contaminative bacteria were identified as Bacillus megaterium and B.aryabhattai,respectively.This is the first report of B.aryabhattai as the contaminative bacterium in sugarcane tissue culture.This study revealed that B.megaterium probably came from endophyte and B.aryabhattai from environment.