Abstract: Objective: To elucidate the structural basis underlying the diverse extremophilic catalytic properties of β-galactosidases in the GH42 glycoside hydrolase family, enzymes with resolved crystal structures were systematically analyzed to reveal the relationship between structural features and catalytic characteristics. Methods: Correlation analysis between catalytic properties and microbial habitats was performed, combined with amino acid composition, phylogenetic evolution, hydrophobicity distribution, and structural bioinformatics approaches, to investigate the structural determinants responsible for catalytic diversity among GH42 β-galactosidases. Results: GH42 β-galactosidases exhibited a biased amino acid composition characterized by relatively high glutamate (E) content and low lysine (K) content, resulting in generally low isoelectric points (pI). The catalytic properties of these enzymes were adaptively correlated with the growth environments of their host microorganisms. A “paired” evolutionary pattern was observed between thermophilic and cold-adapted β-galactosidases within the family. Thermostable enzymes possessed higher internal hydrophobicity, whereas cold-adapted enzymes display stronger surface hydrophilicity. Cold-adapted enzymes exhibited significantly distinct flexible regions at the junction between the TIM-barrel domain and Domain B, as well as at the C-terminus. In contrast, thermostable enzymes enhanced structural rigidity by increasing hydrogen bond numbers and proline (P) content to improve their optimal temperature range. Conclusion: GH42 β-galactosidases share a conserved molecular scaffold and diverge into cold-adapted and thermostable enzymes via differential internal and surface hydrophobicity. Temperature adaptation of these enzymes arises from the synergistic effects of monomer flexibility, number of hydrogen bonds, and oligomeric state. Based on the evolutionary relationships within the family, this study systematically explores the structural divergence and catalytic differentiation among GH42 enzymes. These findings provide a general reference for further investigations into extremophilic catalysis of GH42 β-galactosidases and the development of engineered enzymes with multi-tolerant properties.