Without culture, differentiation of bacille Calmette-Guérin-induced lymphadenitis (BCG-LA) from tuberculosis (TB) is sometimes difficult by histology, but is important because of different treatment schemes. The purpose of this study was to investigate the feasibility of differentiating BCG-LA from TB in lymph nodes (LNs) by molecular analyses of two recently identified genes, pncA and oxyR. In both genes, a single tuberculosis difference exists between Mycobacterium bovis and M. tuberculosis. M tuberculosis complex (MTC) DNA was first detected in nine of ten formalin-fixed, paraffin-embedded LNs from patients aged under 20 years with suspected mycobacterial infections, using polymerase chain reaction (PCR) for IS6110, an insertion sequence specific for MTC species. PCR, together with direct DNA sequencing and PCR-restriction fragment length polymorphism (RFLP) assay, was then performed to identify polymorphic nucleotide in pncA and oxyR, respectively. For comparison, 37 adult cases of tuberculous lymphadenitis were also analysed by PCR-single strand conformation polymorphism (SSCP) assay for pncA and by PCR-RFLP for oxyR. The results revealed that five of the nine IS6110-positive child cases had a G residue at nucleotide 169 in pncA, and also had a three-band pattern after digesting the amplified oxyR segment with AluI, suggesting BCG-LA. The remaining four child cases, as well as all adult cases with detectable IS6110, showed no motility shift in pncA PCR-SSCP and had the same one-band pattern as M. tuberculosis in oxyR PCR-RFLP, suggesting TB lymphadenitis. The data from molecular analyses showed a good correlation with the vaccination history and clinicopathological findings, except for one case. This study indicates that molecular assay of either oxyR or pncA could be a rapid and useful tool to distinguish BCG-LA from TB.