Vitamin B-12 (B-12) production is a strain specific, rare and hidden functional attribute of lactobacilli and a cogent protocol for selection of such isolates from the herd of lactobacilli is required. The present study included isolation of lactobacilli from human samples (milk and fecal), screening them by a polyphasic (three-phase) methodology for probable B-12 production potential and validating the screening protocol by exploring selected strains for in vitro vitamin production (two-phase fermentation) and quantification [micro-assay and ultra fast liquid chromatography (UFLC)]. Fifty-nine Lactobacillus strains were recovered from tested biological samples. Contrary to screening inapplicabilities of first [growth potential (GP) in B-12-free medium] and second phases (GP in B-12-free and cobalt chloride-supplemented conditions), third phase (cbiK gene detection on genomic DNA) alone was revealed as a validated strategy for selection of two probable B-12-producing lactobacilli. Microbiological assay confirmed production and bioavailability of produced vitamin, while UFLC testing validated the results by precisely quantifying the cyanocobalamin (industrially produced bio-available form of B-12) in cell extracts of both possible B12 producers [BHM10 (10.91 +/- 1.55 mu g/l) and BCF20 (23.90 +/- 1.73 mu g/l)] and positive standard [Lactobacillus reuteri DSM20016 (20.03 +/- 4.17 mu g/l)]. Moreover, this study generates a novel report for genomic detection, partial amplification and sequencing of cbiK gene in Lactobacillus plantarum species (both BHM10 and BCF20). In conclusion, contrary to first two phases, cbiK gene detection strategy successfully selects B-12-producing strains from a group of human-originated lactobacilli and can be used in the future for similar screening studies.