A gene probe for the detection of polycyclic aromatic hydrocarbon (PAH) induced nidB and nidA dioxygenase genes has been designed from Mycobacteria JLS, KMS, and MCS. The probe detects a catabolic gene involved in the initial steps of PAH biodegradation in mycobacteria. The gene probe is comprised of three PCR primer sets designed to detect the genes that code for two subunits of the PAH induced dioxygenase enzyme within PAH-degrading mycobacteria. The probe was built by combining three primer sets with a DNA extraction procedure that was designed to lyse the gram-positive mycobacteria cells while in the soil matrix and remove PCR inhibitors. The probe was tested on PAH contaminated soils undergoing bioremediation through landfarming and uncontaminated soils from the same site. The PAH gene probe results demonstrate that the dioxygenase genes can be detected in soils. Sequencing the nidA and nidBPCR products verified that the genes were detected in soil. Comparisons of the sequences obtained from the soil probe to seven known nid gene sequences from various PAH-degrading mycobacteria showed between 97 and 99% nucleotide matches with the nidB gene and 95 and 99% matches with the nidA gene.