Background: The presence in a geographic area of Mycobacterium tuberculosis (Mtb) strains belonging to different phylogeographic lineages and showing different drug susceptibility patterns may suggest recent transmission, with implications in terms of patient clinical management and disease control. The aim of this study was to carry out a preliminary epidemiological investigation of tuberculosis (TB) cases in Rome. Methods: A total of 232 Mtb isolates, collected from new or previously treated patients, admitted between 2008 and 2014 at 2 hospital settings in Rome with a diagnosis of TB, were analyzed by spoligotyping and analyzing 24 variable-number tandem repeats (VNTR) mycobacterial interspersed repetitive-unit (MIRU) loci. The SITVIT2 database and the MIRU-VNTRplus web applications were used to identify the strain genotypes and to generate phylogenetic trees. Results: Based on the position on the phylogenetic tree, 97.4% of the strains were associated with 1 of the 7 main lineages. The Euro-American lineage was the most commonly represented (81.9%) within both Italian and foreign-born populations, although all main lineages were present. The highest frequency of drug-resistant strains was found among the East-Asian lineage (Beijing genotype) isolated from foreign-born patients. Conclusions: Dynamics of TB transmission in Rome indicate recent spread of Mtb strains belonging to phylogeographic lineages and clades usually found in countries and geographic areas with a high incidence of TB, similarly to what is observed in most metropolitan areas in Western Europe. Knowledge from molecular and classical epidemiology provides an important tool for disease control.