Analysis to detect microbes in natural environment requires isolation and subsequent identifications of isolated strain. Success isolation relied on the availability of suitable culture media and growth conditions. Identification mainly based on biochemical tests involving fermentation of carbohydrate substrates, detection of a limited amount of microbial enzymes or other analysis methods. However at most 0.1 to 10% of microscopic ally observed organisms can be grown in the laboratory. Hence many novel microbes, which may be medically and industrial important remains undiscovered. Application of molecular methods has made it possible to identify microorganisms in natural and enrichment samples without the requirements of laboratory cultivation. One such method is whole cell in with hybridization technique based on rRNA sequence analysis. In this study microbial diversity in hot spring has been analyzed by this method. The technology involves genus, species or individual strain specific oligonucleotide probes complementary to rRNA sequences. Successful hybridization leads to identification of the microorganism. Visualization of the microbial cells done by Epifluorescence microscopy.