Gordonia, is a Gram-positive, filamentous bacterial genus within the phylum Actinobacteriathat thrives in diverse environments and exhibits significant catabolic capabilities. These bacteria can degrade heavy metals and hydrocarbons, making them ideal candidates forpotential bioremediation applications. In wastewater treatment plants (WWTPs), Gordoniaspp. are advantageous for mitigating pollutants, aiding in the processing of wastewater. However, the excessive proliferation of Gordonia species, particularly G. amarae, in WWTPs can lead to stable foaming, posing biological, environmental, and operational challenges. Genetic studies of G. amarae may provide insights into their behaviour through the manipulation and transfer of genes between related species. Despite their importance, genetic manipulation of Gordonia species remains challenging due to limited knowledge of plasmids able to replicate within this bacterial genus. This study focuses on plasmids capable of replicating in G. amarae, specifically a large native plasmid pBEN371, from G. pseudoamarae. Gene annotation of pBEN371 revealed the repA and oriV region which enabled the development and use of a shuttle vector for gene cloning in G. amarae CON44Tand related species. This research has expanded our understanding of plasmids that replicate within Gordonia and developed a genetic manipulation toolkit for G. amarae.