Aims: Fermented and probiotic products are mainly based on the activity of diverse Lactobacillus species. Distinguishing of these species, especially the closely related ones might be problematic. Our aim was to compare and evaluate molecular methods that have the potential of discrimination and might serve as alternatives of traditional microbiological techniques. Methods and Results: In our experiments, PCR methods using general and species-specific bacterial primers, RFLP, sequencing and HRM assays were tested and their efficiency compared. A new universal primer pair was designed for amplification of short fragments of the 16S rDNA of six Lactobacillus, a Lactococcus and a Streptococcus species; furthermore, successful HRM analysis was performed on them that resulted in the separation of each species, including the almost indistinguishable Lact. paracasei ssp. paracasei and Lact. paracasei ssp. tolerans subspecies. Conclusions: The results showed that HRM might be a useful, time-and cost-saving one-step tool for preliminary classification of isolates, although the use of additional techniques, like species-specific PCR, analysis of RFLP patterns and sequencing, might be necessary for confirmation of the results. Significance and Impact of the Study: The newly developed HRM primers offer a quick and efficient tool for discrimination of lactobacilli, including very closely related Lactobacillus subspecies.