Tuberculosis (TB) infections in India account for one-third of the global burden, making it important to develop speedy, cost-effective diagnostic tools. This study evaluated recombinant RD1-encoded antigens of Mycobacterium tuberculosis as tools for serodiagnosis by determining the immunological reactivity of these proteins against sera from healthy, bacille Calmette-Guérin (BCG)-vaccinated and TB-infected individuals from Kolkata. Rv3872, Rv3875 (ESAT-6) and Rv3878 were able to discriminate healthy BCG-vaccinated controls from TB patients. Rv3872 showed the highest level of antibody response in comparison with other antigens, and also showed statistically significant differences between pulmonary (p <0.0001) or extra-pulmonary (p <0.001) TB patients and healthy BCG-vaccinated individuals. The levels of antibody were measured using 20-mer overlapping peptides spanning the entire Rv3872 sequence. The immunological reactivity against a mixture of two peptides (P8 and P9) encompassing amino-acids 57-84 correlated well with that obtained using full-length Rv3872. This result was explained by the fact that two of the predicted regions of high antigenicity lie within amino-acid residues 57-85 of Rv3872. The high sensitivity and specificity of Rv3872, as well as the mixture of two synthetic overlapping peptides derived from Rv3872, highlight their potential and argue in favour of their use in serodiagnosis of both pulmonary and extra-pulmonary TB.