<p><i>Cupriavidus necator</i> H16 is the premier lithoautotrophic strain utilized for the industrial bioproduction of polyhydroxyalkanoate (PHA) bioplastics. However, large-scale genetic engineering of this host is hindered by the economic and metabolic burdens of utilizing conventional plasmids that require continuous antibiotic selection. Harnessing the native active partitioning (ParABS) system from the <i>C. necator</i> megaplasmid pHG1 offers a highly promising, selection-free alternative for stable plasmid maintenance. While the <i>trans</i>-acting motor protein (<i>parA</i>) and centromere-binding protein (<i>parB</i>) of pHG1 are annotated, the essential <i>cis</i>-acting centromere sequence (<i>parS</i>) remains unmapped. In this study, we bioinformatically classified the pHG1 operon as a Type Ia ParABS system, noting a distinct lack of homology to six evaluated known <i>parS</i> reference consensus sequences, an evolutionary divergence likely necessary to maintain segregational specificity and prevent plasmid incompatibility within a multipartite genome. To functionally localize the centromeric loci, an in vitro affinity chromatography assay was developed utilizing an immobilized, recombinant N-terminal hexahistidine-tagged ParB (H₆ParB). The assay demonstrated the specific retention of a ~ 2.7&#xa0;kb PAR fragment located immediately downstream of the <i>parAB</i> operon, that indicating the regional presence of the primary <i>parS</i> locus. Furthermore, the assay indicated a putative, secondary lower-affinity ParB-binding site within an upstream ~ 4.6&#xa0;kb ORI fragment. While future in vivo validation and high-resolution sequencing are required to precisely map the exact palindromic motif, isolating the continuous PAR region identifies a complete and self-contained partitioning module. This localization establishes the necessary biochemical foundation for engineering robust that selection-free expression vectors tailored for industrial PHA bioproduction.</p>