Soil samples were collected from oil-contaminated sites which were located in west Qurna, Basrah, Iraq. Pseudomonas species were initially isolated on mineral salts and Pseudomonas agar media and identified using morphological and biochemical characterizations. Then, specific primers for the rhlA gene belonging to Pseudomonas aeruginosa were designed based on the primer design conditions, and PCR was performed to amplify the 888 bp size fragment of the rhlA gene; additionally, the primary PCR products were purified and sent for sequencing. The band of about 888bp was determined on the gel, the amplified rhlA gene sequencing findings were revised, only 366 bp were ready to analyze using the (BLAST) software, and the final result was identified as a partial sequence of chromosomal rhlA gene related to Pseudomonas aeruginosa with percent identity of 99.45%. The query gene’s incomplete matching with another partial rhlA record on NCBI was caused by variations in two base pair sequences (T in sequence 348 and C in sequence 353, respectively), and despite the small difference, this results in variation in the amino acids produced; so that a new record number, ON637169, was assigned when the sequence was deposited in GenBank. The relation among the new record of partial rhlA gene with the same number of the other rhlA gene sequences (60 records) was demonstrated by creating a phylogenetic tree.