Transcription factors are a family of proteins that control the physiological and biochemical process of a plant. The MYB family of proteins is the largest family of transcription factors (TF) that plays an important role in controlling growth and development and stress responses. These genes have been extensively studied in many dicot and monocot plants. Among the grasses, sugarcane has a complex polyploidy genome, and only little information is available about the MYB TF family. With increasing importance of this biofuel crop, 57 R2R3-MYB, 20 MYB-like, and 1 3R1-MYB genes were computationally predicted and 51 ScMYBs were isolated by RT-PCR. Subgroup-specific conserved motifs outside the MYB domain demonstrated their phylogeny and functional conservation in sugarcane. Based on their biological process, the isolated ScMYB genes were functionally categorized into cell growth and maintenance (39.74 %), stress response (33.33 %), metabolism (14.10 %), and developmental regulation (12.82 %). Expression analysis by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) revealed a significantly higher expression trend of ScMYB26/AS1 > ScMYB54 > ScMYB66 > ScMYB75 transcripts in response to water deficit, whereas in salt stress, ScMYB1 and ScMYB58 showed higher expression during 12 days of stress (DOS) in sugarcane leaves. Further, the stress-responsive ScMYB26/AS1 was functionally characterized by agroinfiltration-mediated transient expression in tobacco. Semi-qRT-PCR demonstrated the increased expression of introduced ScMYB26/AS1 transcript in transformed tobacco explants during stress induction. Taken together, our comparative genomics analysis and isolation of ScMYB genes from sugarcane illustrated the viable, future solid foundation for improvement of agricultural crops to stress tolerance by a transgenic approach.