To investigate the molecular epidemiological characteristics of Mycobacterium tuberculosis strains collected in Gifu Prefecture, Japan, 483 M. tuberculosis clinical isolates were used for Japan Anti-Tuberculosis Association (JATA) 18-variable number tandem repeats (VNTR) analysis during 2015–2019. To estimate the lineages of M. tuberculosis strains, JATA18-VNTR profiles were applied to a maximum a posteriori method. The results revealed that the ancient Beijing subfamily, accounting for 57.3% (277/483) was the most prevalent M. tuberculosis strain. Furthermore, 18 clusters (GC-1–GC-18) were found by minimum spanning tree analysis. The proportion of clustering strains was 9.9% (48/483), and epidemiological links to these clusters were unclear without GC-6 and GC-18. Meanwhile, interestingly, VNTR profiles of GC-7–GC-9 and GC-14 were indistinguishable from the regional epidemic strains of Nagoya City, which has a strong socioeconomic relationship with Gifu Prefecture, but did not match the nationwide epidemic strains. This study suggests that coordinated analyses within prefectures with strong socioeconomic relationships are important.