We sequenced the entire tb1 gene in six maize inbreds and its wild relatives (parviglumis, mexicana, perennis and luxurians) to characterize it at molecular level. Hopscotch and Tourist transposable elements were observed in the upstream of tb1 in all maize inbreds, while they were absent in wild relatives. In maize, tb1 consisted of 431–443 bp 5′UTR, 1101 bp coding sequence and 211–219 bp 3′UTR. In promoter region, mutations in the light response element in mexicana (~ 35 bp and ~ 55 bp upstream of TSS) and perennis (at ~ 35 bp upstream of TSS) were found. A 6 bp insertion at 420 bp downstream of the polyA signal site was present among teosinte accessions, while it was not observed in maize. A codominant marker flanking the 6 bp InDel was developed, and it differentiated the teosintes from maize. In Tb1 protein, alanine (12.7–14.6%) was the most abundant amino acid with tryptophan as the rarest (0.5–0.9%). The molecular weight of Tb1 protein was 38757.15 g/mol except ‘Palomero Toluqueno’ and HKI1128. R and TCP motifs in Tb1 protein were highly conserved across maize, teosinte and orthologues, while TCP domain differed for tb1 paralogue. Tb1 possessed important role in light-, auxin-, stress-response and meristem identity maintenance. Presence of molecular signal suggested its localization in mitochondria, nucleus and nucleolus. Parviglumis and mexicana shared closer relationship with maize than perennis and luxurians. A highly conserved 59–60 amino acids long bHLH region was observed across genotypes. Information generated here assumes significance in evolution of tb1 gene and breeding for enhancement of prolificacy in maize.