At present, the Lactobacillus fermentum 90 TC-4 strain is widely used for production of probiotics, dietary supplements, and food. It is for this reason that it is topical to study this strain using modern molecular genetic methods. The biochemical properties of the strain were examined using the API 50 CHL test system (BioMerieux, France), genome sequencing was carried out using the MiSeq (Illumina) platform, and de novo genome assembly was performed using the Spades, MIRA 4.0, and Newbler 2.6 software. Genome annotation was carried out with the help of the Prokka v. 1.11 utility and RAST and BASys genomic servers. The main characteristics of the L. fermentum 90 TC-4 genome were established. It was proven that the genome does not have any determinants of pathogenicity, virulence or antibiotic resistance. It was shown that the low saccharolytic ability of the strain is associated with the absence of appropriate transport systems—the sucrose-specific phosphonolpyruvate system PTS_ScrA and ribose-specific RbsD permease—as well as several enzymes. The CRISPR-Cas locus of the strain was analyzed, unique spacers (which in future can be used for strain indication) were revealed, and molecular mechanisms of antibiotic resistance of the strain were studied. Using the MLST scheme presented in the scientific literature, allelic profiles of housekeeping genes were established. It was also found that the allelic profile obtained for the L. fermentum 90 TC-4 strain does not correspond to any of the previously described sequence types. Since L. fermentum 90 TC-4 does not have determinants of antibiotic resistance, pathogenicity, virulence, or integrated plasmids, the strain does not pose any hazard in terms of spread of these determinants and can be used as a probiotic producer strain. The observed features of the CRISPR locus and allelic profile can further be used to indicate the strain.