Lactic acid bacteria (LAB) are widely used in various sectors of food industry, in biotechnology and in medicine, therefore it is very important to properly identify them and to correctly assess their intra-species differentiation. The selection of appropriate molecular analysis techniques should take into account the high accuracy, repeatability and typability of the method. The objective of the research study was to assess the possibility of differentiating 12 Lactobacillus strains with the use of techniques commonly applied in laboratories: the sequencing of the 16S  rDNA gene, pheS gene and MALDI-TOF MS. Based on the analysis results obtained, it was found that in the tested strains the pheS gene was characterised by a high level of homology (98 %)  and a low discriminant power. In the two independent MALDI-TOF MS analyses the same result was obtained for 10 strains: seven – L. brevis (DSM 6235, 102, 103, 489, 863, 975, 3/16/1), two – L.  plantarum (1178, 133) and one – L. curvatus 557. After the performed sequencing analysis of the 16S rDNA gene the findings were confirmed only in the case of five (DSM 6235, 3/16/1 and 489) of  the twelve strains tested. The comparison of the results obtained made it possible to conclude that as for the selected LAB strains the highest differentiating value had the analysis based on the  sequencing of the 16S rDNA gene.