ParB proteins are one of the three essential components of partition systems that actively segregate bacterial chromosomes and plasmids. In binding to centromere sequences, ParB assembles as nucleoprotein structures called partition complexes. These assemblies are the substrates for the partitioning process that ensures DNA molecules are segregated to both sides of the cell. We recently identified thesopCcentromere nucleotides required for binding to the ParB homologue of plasmid F, SopB. This analysis also suggested a role insopCbinding for an arginine residue, R219, located outside the helix-turn-helix (HTH) DNA-binding motif previously shown to be the only determinant forsopC-specific binding. Here, we demonstrated that the R219 residue is critical for SopB binding tosopCduring partition. Mutating R219 to alanine or lysine abolished partition by preventing partition complex assembly. Thus, specificity of SopB binding relies on two distinct motifs, an HTH and an arginine residue, which define a split DNA-binding domain larger than previously thought. Bioinformatic analysis over a broad range of chromosomal ParBs generalized our findings with the identification of a non-HTH positively charged residue essential for partition and centromere binding, present in a newly identified highly conserved motif. We propose that ParB proteins possess two DNA-binding motifs that form an extended centromere-binding domain, providing high specificity.