Ferulate 5-hydroxylase (F5H), a cytochrome P450-dependent monooxygenase, catalyzes the hydroxylation of coniferaldehyde, a crucial step in the formation of syringyl lignin monomer (S). However, evolutionary divergence, expression patterns under abiotic stress conditions (ABA, PEG and NaOH) and lignin content-related features of the F5H gene family in Sorghum bicolor have not been explored. This study envisaged mining of Sorghum genomic data leading to the identification of 61 SbF5H genes. Bioinformatics analysis revealed the phylogenetic evolutionary relations, gene structures, conserved motifs, physicochemical properties, and promoter cis-acting elements related to these genes and their encoded proteins. Based on the gene structural and phylogenetic features, these 61 SbF5Hs were grouped into 4 subclasses. The in silico expression analysis revealed higher accumulation of SbF5H1 transcripts in embryo and in root under stress conditions. Similarly, Other SbF5H genes have shown expression in stem and root, thus indicating SbF5H genes involvement in Sorghum lignin biosynthesis. By exploring into the functional aspects of the F5H gene, our study sought to shed light on its significance in influencing not only the chemical makeup of lignin but also the resultant plant phenotypes. This insight into the molecular mechanisms governing lignin biosynthesis can have implications for bioenergy production and crop improvement.