β -D-Galactosidases (EC 3.2.1.23) hydrolyze the terminal nonreducing β -D-galactose residues in β -D-galactosides and are ubiquitously present in all life forms including extremophiles. Eighteen microbial β -galactosidase protein sequences, six each from psychrophilic, mesophilic, and thermophilic microbes, were analyzed. Primary structure reveals alanine, glycine, serine, and arginine to be higher in psychrophilic β -galactosidases whereas valine, glutamine, glutamic acid, phenylalanine, threonine, and tyrosine are found to be statistically preferred by thermophilic β -galactosidases. Cold active β -galactosidase has a strong preference towards tiny and small amino acids, whereas high temperature inhabitants had higher content of basic and aromatic amino acids. Thermophilic β -galactosidases have higher percentage of α -helix region responsible for temperature tolerance while cold loving β -galactosidases had higher percentage of sheet and coil region. Secondary structure analysis revealed that charged and aromatic amino acids were significant for sheet region of thermophiles. Alanine was found to be significant and high in the helix region of psychrophiles and valine counters in thermophilic β -galactosidase. Coil region of cold active β -galactosidase has higher content of tiny amino acids which explains their high catalytic efficiency over their counterparts from thermal habitat. The present study has revealed the preference or prevalence of certain amino acids in primary and secondary structure of psychrophilic, mesophilic, and thermophilic β -galactosidase.