β-galactosidase (Lactase), an enzyme belonging to the glycoside hydrolase family causing the hydrolysis and trans-glycosylation ofβ-D-galactosides, has a vital role in dairy industries. The current investigation emphasizes onin-silicoidentification and comparative analysis of different fungal lactases present inAspergillus fumigatus,Aspergillus oryzae,Botrytis cinerea, andFusarium 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 ofβ-galactosidases. Evolutionary and homology relationships were established by phylogenetic and synteny analyses. A total of 14β-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 betweenAspergillus fumigatusandAspergillus oryzae. Structural insights allowed the prediction of best protein models based on the higher ERRAT and Q-MEAN values. And RNA-sequencing analysis, performed onA.fumigatus, elucidated the role ofβ-gal in germ tube development. This study would pave the way for efficient fungal lactase production as it identifiedβ-gal genes and predicted their various features and also it would provide a road-way to further the understanding ofA.fumigatuspathogenicity via the expression insights ofβ-gal in germ tube development.