RAPD conditions were optimized to generate reproducible banding patterns by testing primers, thermocyclers and overall reproducibility in repeat DNA analysis and separate DNA extractions. Five primers were chosen on the basis of band intensity and distribution (between 2 and 10 bands) which clearly distinguished among strains of Bifidobacterium adolescentis, B. animalis, B. bifidum, B. breve, B. infantis and B. longum. The use of five single-primer reactions under optimized conditions improved the resolution and accuracy of the RAPD method for the characterization of dairy-related bifidobacteria. The results indicated that this method was highly reproducible in repeated analysis. Similarity between bifidobacteria strains was evaluated based on their RAPD profiles. Using a set of five primers, it was demonstrated that it may be possible to distinguish three different species of Bifidobacterium (B. breve, B. bifidum and B. adolescentis), based on similarity of the RAPD profiles to known reference strains. Furthermore, application of the RAPD technique may also be useful and faster, than traditional systematics for placement of industrial strains into specific clusters (either B. longum/infantis or B. animalis/lactis).