Aims: A molecular methodology based on PCR-associated automated ribotyping was developed to specifically detect the Lactobacillus strains of two probiotic products (an orally administered lyophilized preparation and vaginal tablets) in human faeces and vaginal swabs. Methods and Results: The 16S-23S rDNA sequences and the ribotype profiles of the probiotic lactobacilli were characterized and new species-specific primer sets were designed. The identification of faecal and vaginal lactobacilli isolated from subjects administered with the probiotic products was performed by using PCR with species-specific primers followed by strain-specific automated ribotyping. Conclusions: The PCR-ribotyping identification allowed to study the colonization patterns of the probiotic lactobacilli in the human gut and vagina evidencing the strains with the best survival capability. Significance and Impact of the Study: The proposed molecular method represents a powerful tool of strain-specific identification, useful for differentiating exogenous from indigenous strains in any microbial ecosystem and for rationally choosing probiotic bacteria with the best chance of survival in the host.