The development of a direct faecal PCR (DPCR) test for both ovine JD and bovine JD 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 applicable mainly to research applications because of its complexity and labour intensity. Here we report the development, optimisation and performance of the HT-J test for the detection Johne’s disease in cattle and sheep. To validate the HT-J test, 1329 cattle and 596 sheep faecal samples, representing unexposed and exposed herds and flocks, were tested in two laboratories. The new test has demonstrated sensitivity and specificity equal to or greater than culture and is suitable for use in routine diagnostic laboratories. The HT-J test was developed to suite 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.