The cis-acting P1 and P7 parS sites are responsible for active partition of P1 and P7 plasmids to daughter cells. The two sites are similar but function only with ParB proteins from the correct species. Using hybrid ParB proteins and hybrid parS sites, we show that specificity is determined by contacts between bases that lie within two parS hexamer boxes and a region in the ParB C-terminus. We refer to these contacts as discriminator contacts. The P7 discriminator contacts were mapped to 3 and 2 bp respectively within the two parS hexamer boxes, and a 10 amino acid region of P7 ParB. Similarly placed residues of different sequence are responsible for the P1 discriminator contact. The discriminator contacts are distinct from previously identified DNA binding contacts which involve different ParB and parS regions. Deletion of the ParB C-terminus that makes the discriminator contact does not diminish in vitro binding but renders it species independent. The discriminator contact is therefore a negative function, interfering with binding of the wrong ParB, but not providing energy for the binding of the correct one. Similar discriminator contacts might be responsible for specificities seen among families of eukaryotic DNA binding proteins that share common binding motifs.