Cancer chemotherapy can lead to the mycobacterial infections. Tuberculosis has been reported a serious complication in leukemia patients who undergo chemotherapy. The study was focused to find mutations in hupB gene of M. tuberculosis in 50 acute lymphoblastic leukemia (ALL) patients through semi multi complex PCR. A column based DNA isolation method was adopted for DNA isolation. The gene for histone-like protein (hupB [Rv2986c]) of M tuberculosis was amplified to detect two closely related mycobacterial species. Primers M and S (histone like protein HupB) were utilized to generate amplicons of 318 bp and 291 bp for M. tuberculosis and M. bovis, respectively. Out of fifty ALL patients, 21 (42%) were females and 29 (58%) were males. The prevalence of ALL was found higher in males as compared to females. The prevalence of ALL was higher in patients of age group 5-10 years. The results of the amplification showed that, the 318 bp fragment specific for M. tuberculosis was observed in seven samples (14%), while 291 bp fragment specific for M. bovis was not observed in any sample. Children with ALL were found at higher risk for tuberculosis. A risk evaluation of tuberculosis infection must be conducted before managing chemotherapy.