ABSTRACT Amino acid sequences of β-galactosidase enzyme belonging to different families of bacteria, fungi and plants retrieved from GenPept database were analyzed for multiple sequence alignment, cluster analysis, conserved motif discovery and their Pfam analysis using different bioinformatics tools. The multiple sequence alignment revealed different conserved residues of amino acids exclusively for each groups except fungi. The cluster analysis for different groups uniformly showed three major clusters based on the closeness of the β-galactosidase protein sequences irrespective of the source organisms. Seven conserved motifs belonging to different families were assessed. These identified motifs showed the evolutionary closeness among species at the molecular level. KEYWORDS β -galactosidase, conserved motif, cluster analysis, residues For More Details: https://wireilla.com/papers/ijbb/V3N2/3213ijbb04.pdf