Abstract: The present study reports on the isolation and characterization of new isolates from crude oil from petroleum contaminated soil. The isolates were then screened for their potential towards biodegradation of toluene by growing them in both solid as well as liquid mineral medium supplemented with toluene as a sole carbon source and further identified by GC-FAME analysis. The degradation potential of these isolates strongly suggests that the isolates have TOL like plasmid and carries xylM gene involved in the expression of the toluene monooxygenase. The primer sets were used in PCR to assess the presence of the catabolic gene in new toluene degrading isolates. The primer verification was done by using bioinformatics tools. From the results it is conclusive that isolates were able to degrade Toluene, and from PCR it is evident that the isolates contained gene coding for Toluene degradation. The test results indicate that the bacteria could contribute to bioremediation of aromatic hydrocarbon pollution.