In the present study, probiotic, safety and functional characteristics of eight indigenous bifidobacterial isolates were compared to identify suitable strains for functional food application. Among the isolates, six strains of Bifidobacterium longum and one each of Bifidobacterium breve and Bifidobacterium bifidum were identified by 16S rRNA, xfp and hsp60 gene sequencing. Diversity among these strains was established by RAPD and Rep-PCR. Genes associated with sortase-dependent pili (SDP) (credited for role in adhesion) and serpin (immunomodulation) which can serve as potential marker genes for rapid identification of probiotic Bifidobacterium, was also evaluated. All the isolates exhibited potential probiotic, functional (antimicrobial activity, antioxidant activity, phytase activity, milk fermentation ability) and safety attributes. However, among them, B. breve NCIM5671 exhibited, better tolerance to low pH, amylase activity and exopolysaccharide producing ability. B. bifidum NCIM5697 and B. longum NCIM5672 demonstrated higher adherence ability to Caco-2 cells. NCIM5697 also displayed exopolysaccharide producing ability while NCIM5672 showed strong antibacterial activity against pathogens tested. Further, with respect to presence of adhesion marker genes, disparity was observed among B. longum strains. B. longum NCIM5684 and B. longum NCIM5686 displayed presence of subunits of SDP reported to be present in B. breve. In addition, B. longum NCIM5686 also lacked SDP present in all other B. longum isolates. B. breve NCIM5671, B. longum NCIM5672 and B. bifidum NCIM5697 with appreciable traits qualifies as potential probiotic cultures. Further, the variations observed in molecular and functional characteristics of isolates signify genetic diversity among the cultures.