The objective of our investigation was to develop a convenient and reliable method of generating template DNA for routine PCR-based detection and typing of lactic acid bacteria (LAB). Template DNA extracted from Lactobacillus, Lactococcus, Pediococcus and Leuconostoc using a combination of urea, SDS and NaOH yielded amplicons of expected size in PCR with genus-specific primers. Apart from LAB, the proposed method could also be adopted to generate PCR-compatible template DNA from a number of Gram-positive and Gram-negative bacterial strains. DNA template prepared by the proposed method from various standard strains of Lactobacillus sp. also generated discriminating fingerprints with BOXA1R primer in rep-PCR. A significant finding of the investigation was that a comparable banding profile of LAB strains was obtained in rep-PCR using template DNA prepared by urea-SDS-NaOH method and a commercially available DNA isolation kit. This was further evidenced by high dice coefficient values obtained in the range of 81.8-96.7 when cluster analysis was performed by UPGAMA method. The application potential of this DNA extraction method for PCR-based direct detection of LAB in fermented food samples such as dahi, idli batter and salt-fermented cucumber was validated by detecting specific amplicons of LAB genera in the fermented samples. The applicability of the proposed template DNA extraction method was further substantiated when 29 bacteriocinogenic LAB strains (Bac+) previously detected in salt-fermented cucumber by PCR [Singh, A.K., Ramesh, A., 2008. Succession of dominant and antagonistic lactic acid bacteria in fermented cucumber: Insights from a PCR-based approach. Food. Microbiol. 25, 278-287] generated differentiating fingerprints in BOX element based rep-PCR and formed clusters with reference LAB strains.