Aim:  Molecular cloning, overexpression and biochemical characterization of the genes from the Mycobacterium tuberculosis H37Rv genome having hypothetical β‐lactamases activity. Methods and Results:  Analysis of the M. tuberculosis H37Rv genome revealed that Rv2068c, Rv0406c and Rv3677c gene products were predicted to exhibit β‐lactamases activity. All the three genes were cloned in pET28a vector and overexpressed in C41 (DE3) Escherichia coli cells. The His‐tagged recombinant proteins were confirmed by immunoblotting and were shown to have β‐lactamase activity by the hydrolysis of nitrocefin and other β‐lactams. Catalytic parameters for all the recombinant proteins were derived followed by the enzyme inhibition studies. Antibiotic susceptibility studies using the recombinant strains showed an increased resistance against different classes of β‐lactam antibiotics. Conclusion:  The study revealed the possibility of more than one gene in M. tuberculosis, encoding proteins having β‐lactamase or β‐lactamase‐like activity, giving wide spectrum of resistance against β‐lactams. Significance and Impact of the Study:  Systematic study of hypothetical β‐lactamases of M. tuberculosis and related species and their correlation with β‐lactam and inhibitor susceptibility profile might be useful in developing new antibiotic regime for the treatment of tuberculosis caused by multiple drug resistant (MDR) strains.