Spoligotyping was applied to old (5-11 years) Ziehl-Neelsen (ZN)-stained smears for strain identification and differentiation and to predict the utility of the technique in epidemiological studies. Among 57 DNA samples extracted from ZN slides lying stored at room temperature, 93% (53) amplification was achieved for mpt64 gene. Spoligopatterns were generated from 77.7% (41/53) DNA samples, whereas negative controls did not yield any spoligopatterns. All slides with 2+ (n=20) and 3+ (n=13) positivity while 42% (11/26) of slides with low positivity (< or 1+) showed a good signal and a reproducible pattern. This technique may have application in identification of spoligotypes in control programme implemented areas remote from research laboratory and would also increase our knowledge about the clonal structure of Mycobacterium tuberculosis in the population, when applied to old samples in different locations.