Background: Tuberculosis is an important cause of death with an increased antibiotic resistance. Kanamycin and amikacin are among the most effective aminoglycosides for multidrug-resistant strains. We investigated the resistance to these two antibiotics in 111 susceptible and 9 resistant specimens. Materials and Methods: Phenotypic method was used to assess the resistance, PCR method was performed for rrs and eis genes and the results confirmed by sequencing. Results: No resistance against kanamycin and amikacin was observed in susceptible specimens and only three resistant samples had such resistance. Two had mutations at A1401G nucleotide, and the nucleotide change of G→A was observed at 1484 position in one sample. Mutations at A514C, A907C, G1332A positions were observed in one sample. eis gene sequencing revealed that one sample had mutation in promoter region at A-13G position. Conclusion: The most important mutation was at nucleotide A1401G in rrs gene and a low prevalence of aminoglycoside resistance was observed in northeastern Iran.