β-galactosidase (Lactase), an enzyme belonging to the glycoside hydrolase family causing the hydrolysis and trans-glycosylation of β-D-galactosides, has a vital role in dairy industries. The current investigation emphasizes on in-silico identification and comparative analysis of different fungal lactases present in Aspergillus fumigatus, Aspergillus oryzae, Botrytis cinerea, and Fusarium fujikuroi. Prediction of motifs and domains, chromosomal positioning, gene structure, gene ontology, sub-cellular localization and protein modeling were performed using different bioinformatics tools to have an insight into the structural and functional characteristics of β-galactosidases. Evolutionary and homology relationships were established by phylogenetic and synteny analyses. A total of 14 β-gal genes (GH-35) were identified in these species. Identified lactases, having 5 domains, were predicted to be stable, acidic, non-polar and extracellularly localized with roles in polysaccharide catabolic process. Results showed variable exonic/intronic ratios of the gene structures which were randomly positioned on chromosomes. Moreover, synteny blocks and close evolutionary relationships were observed between Aspergillus fumigatus and Aspergillus oryzae. Structural insights allowed the prediction of best protein models based on the higher ERRAT and Q-MEAN values. And RNA-sequencing analysis, performed on A. fumigatus, elucidated the role of β-gal in germ tube development. This study would pave the way for efficient fungal lactase production as it identified β-gal genes and predicted their various features and also it would provide a road-way to further the understanding of A. fumigatus pathogenicity via the expression insights of β-gal in germ tube development.