Understanding the mechanisms of Cu pollution-induced community tolerance (PICT) in soil requires the characterization of Cu-resistant microorganisms at a community level using modern molecular tools. A primer pair (copAF2010 (5 -TGCAC CTGAC VGGSC AYAT-3 )/copAR2333 (5 -GVACT TCRCG GAACA TRCC-3 )) tentatively targeting Pseudomonas-like Cu-resistant microorganisms was designed in this study. The specification of the primers was tested through conventional polymerase chain reaction (PCR) and the construction of a Pseudomonas-like copA gene fragment library, and then the primers were used to quantify the Cu-resistant microorganisms using quantitative PCR technique. A significant increase of Cu-resistant microorganisms targeted by the primers was observed in a paddy soil from Jiaxing, China which has been exposed to one-year Cu contamination. The results provided direct evidence for Cu PICT in the soil, and the quantification method developed in this study has the potential to be used as a molecular assay for soil Cu pollution.