Background: The MBF1 genes are transcription co-activators that trigger the transcription factors activity by bridging TATA-element binding proteins and basic leucine zipper (bZIP) transcription factors in yeast, human and drosophila. MBF1s activate the gene expression in response to plant development, abiotic stresses including heat, cold, salt, drought and biotic stress, including pathogen attack. It also plays a critical role in physiological and biochemical processes, including hormonal regulation, lipid metabolism and cell differentiation. However, MBF1's role in cotton is still unknown. Results: The multiprotein bridging factor1 (MBF1) genes are being characterized for the first time in four cotton genomes. A total of 24 MBF1 genes were identified in four cotton genomes, including 8 genes in G. hirsutum (GhMBF01-GhMBF08), 8 in genes G. barbadense (GbMBF01-GbMBF08), 4 genes in G. arboreum (GaMBF01-GaMBF04) and 4 in G. raimondii (GrMBF01-GrMBF04) respectively. Phylogenetic relationship analysis in 11 plant species suggested the classification of MBF1 genes into two major groups that suggested evolutionary uniqueness in both groups. Comparative analyses of structural features, motif conservation, cis-regulatory elements, genes enrichment and protein-protein interaction network suggested distinctive characteristics, functions and their relation with biologically functional genes. A comparative transcriptome analysis proposed the role of MBF1 genes in plant growth, response to abiotic and biotic stress tolerance, seed germination and cotton fiber development. The qRT-PCR validation of GhMBF02 (GH_A04G0454, Type b), GhMBF04 (GH_A08G2678, Type b), GhMBF07 (GH_D06G0632, Type C), and GhMBF08 (GH_D08G2673, Type b) suggested a positive role of MBF1 in fiber elongation stages of fiber quality specific RIL population lines. Conclusion: Present study introduced and provided useful information about the potential of MBF1 family genes in response to various growth and developmental stages, biotic and abiotic stresses in cotton that can be referred for further function-based experiments.