Agrobacterium vitis, the causal agent of grape crown gall, can have severe economic effects on grape production. The bacterium survives systemically in vines and, therefore, is disseminated in propagation material. We developed an assay for use in indexing programs that is efficient and sensitive for detecting A. vitis in grape tissue. Initially, real-time polymerase chain reaction (PCR) primers specific for diverse tumorigenic strains of A. vitis were developed using the virD2 gene sequence. To overcome the effects of PCR inhibitors present in plant tissue, DNA extraction methods that included magnetic capture hybridization (MCH), immunomagnetic separation (IMS), and extraction with the Mo Bio Powerfood kit were compared. The assays incorporating MCH or IMS followed by real-time PCR were 10,000-fold more sensitive than direct real-time PCR when tested using boiled bacterial cell suspensions, with detection thresholds of 10(1) CFU/ml compared with 10(5) CFU/ml. DNA extraction with the Powerfood DNA extraction kit was 10-fold more sensitive than direct real-time PCR, with a detection threshold of 10(4) CFU/ml. All three assays were able to detect A. vitis in healthy-appearing grapevine cuttings taken from infected vines.