The development of a direct faecal polymerase chain reaction (DPCR) test for both ovine Johne's disease and bovine Johne's disease has been a goal in Johne's disease research since the mid 1990s. We have developed a new high throughput DPCR test (termed the HT-J test), suitable for faecal samples from both cattle and sheep. The HT-J test is based on a highly sensitive DPCR test that was useful for research applications but not suitable as a routine diagnostic test because of its complexity and labour intensity (Kawaji et al., 2007). The HT-J test was developed to suit Australian conditions and, during the course of the study, it became apparent that local optimisation is paramount to the success of the test. Furthermore, sample storage prior to testing was identified as a critical issue. To validate the HT-J test, 1,585 bovine faecal samples, representing 31 individual submissions from both unexposed and exposed herds, were tested in two laboratories. Here we report the development, optimisation and performance of a new high throughput DPCR test for the detection Johne's disease in cattle and sheep. The new test has demonstrated sensitivity and specificity equal to or greater than culture and is suitable for use in routine diagnostic laboratories.