In 2018, a few sequencing studies were published revealing the existence of two monophyletic clusters within the C. diphtheriae species, meaning that this species can be divided into two subspecies: C. diphtheriae subsp. diphtheriae and C. diphtheriae subsp. lausannense. The objective of our study was to describe the genotype and the phenotype of 2 nontoxigenic C. diphtheriae strains isolated in Russia in 2017–2018, which were classified by us as C. diphtheriae subsp. lausannense based on the aggregated data yielded by a variety of techniques, including microbiological and molecular genetic techniques, as well as a bioinformatic search for subspecies-specific genes in the publicly available genomes of C. diphtheriae. The isolated strains had morphological and biochemical characteristics of C. diphtheriae. The strains were assigned to the MLST type ST199 included in the clonal complex associated with subsp. lausannense. PCR revealed that both analyzed strains of C. diphtheriae subsp. lausannense carried the ptsI gene encoding phosphoenolpyruvate-protein phosphotransferase and did not carry the narG gene encoding the synthesis of nitrate reductase subunits, whereas the strains of C. diphtheriae subsp. diphtheriae had the narG gene and did not have ptsI. We experimentally proved the ability of lausannense strains to ferment N-acetylglucosamine. Our findings expand the knowledge of the biological diversity of C. diphtheriae and indicate the need for estimating the spread of these microorganisms in Russia, as well as their pathogenic potential.