We investigated the lineages of Mycobacterium tuberculosis (Mtb) isolates from the RYOKEN study in Japan in 2007 and the usefulness of genotypic drug susceptibility testing (DST) using the Genome Research for Asian Tuberculosis (GReAT) database. In total, 667 isolates were classified into lineage 1 (4.6%), lineage 2 (0.8%), lineage 2/Beijing (72.1%), lineage 3 (0.5%), and lineage 4 (22.0%). The nationality, gender, and age groups associated with the isolates assigned to lineage 1 were significantly different from those associated with other lineages. In particular, isolates of lineage 1.2.1 (EAI2) formed sub-clusters and included a 2,316-bp deletion in the genome. The proportion of the isolates resistant to at least one anti-tuberculosis (TB) drug was 10.8%, as determined by either the genotypic or phenotypic method of DST. However, the sensitivities to isoniazid, streptomycin, and ethambutol determined by the genotypic method were low. Thus, unidentified mutations in the genome responsible for drug resistance were explored, revealing previously unreported mutations in the katG, gid, and embB genes. This is the first nationwide report of whole-genome analysis of TB in Japan.