Recently, a novel species, Mycobacterium yongonense (DSM 45126T), was introduced and while it is phylogenetically related to Mycobacterium intracellulare, it has a distinct RNA polymerase β-subunit gene (rpoB) sequence that is identical to that of Mycobacterium parascrofulaceum, which is a distantly related scotochromogen, which suggests the acquisition of the rpoB gene via a potential lateral gene transfer (LGT) event. The aims of this study are to prove the presence of the LGT event in the rpoB gene of the M. yongonense strains via multilocus sequence analysis (MLSA). In order to determine the potential of an LGT event in the rpoB gene of the M. yongonense, the MLSA based on full rpoB sequences (3447 or 3450 bp) and on partial sequences of five other targets [16S rRNA (1383 or 1395 bp), hsp65 (603 bp), dnaJ (192 bp), recA (1053 bp), and sodA (501 bp)] were conducted. Incongruences between the phylogenetic analysis of the full rpoB and the five other genes in a total of three M. yongonense strains [two clinical strains (MOTT-12 and MOTT-27) and one type strain (DSM 45126T)] were observed, suggesting that rpoB gene of three M. yongonense strains may have been acquired very recently via an LGT event from M. parascrofulaceum, which is a distantly related scotochromogen.