Over time and concurrent development of methods to identify and characterise bacteria, the lactic acid bacteria (LAB) have undergone multiple taxonomic revisions. As a result of the revisionary nature of LAB taxonomy, the historical characterisation of Lactobacillus acidophilus has struggled with misidentification and misrepresentation. Now however, due to its global use in food products for both flavour and probiotic effect, L. acidophilus is now one of the most well physiologically characterised Lactobacillus species. Bifidobacterium bifidum and Bifidobacterium animalis subsp. lactis are also LAB that are considered to have probiotic effects. Here modern high-throughput next generation comparative genomic techniques are used alongside conventional biochemical and molecular typing methods to analyse the sub-species level diversity of these three probiotic species. Results Randomly Amplified Polymorphic DNA (RAPD) profile similarity analysis showed limited strain-level diversity of L. acidophilus. A species specific marker test was developed for L. acidophilus and used to search for L. acidophilus in wild rodent and human faeces. No L. acidophilus was detected in wild rodent faeces and its carriage in human faeces was highly variable. High-throughput next generation sequencing was used to resequence the genomes of 28 L. acidophilus isolates. Comparing these sequences indicated a high level of genomic conservation in L. acidophilus, which was reflected by limited phenotypic diversity. Comparative genomics in Bifidobacterium animalis subsp. lactis supported the hypothesis that it is a clonally monophyletic species, whereas B. bifidum strains were genomically diverse. Conclusions Methods for phenotypically characterising and typing LAB have generally been superseded in accuracy by DNA sequence based methods. Probiotic bacteria display a range of subspecies level population structures. Commercial and culture collection L. acidophilus isolates did not significantly differ phenotypically, but were distinct when their genome sequences are compared. B. bifidum was genotypically diverse at the subspecies level, while B. animalis subsp. lactis appeared to be clonally monophyletic. Comparative genomics and genome (re)sequencing of probiotic bacteria will become a “gold standard” method for characterisation and typing of isolates.