Karnal bunt (Tilletia indica), the most important biosecurity and market access threat to the Australian wheat industry, can be seed, soil and air borne. The spores accumulate in black masses in the spikes of infected plants and are released in the air and dust fractions during harvest, transport, and storage operations. Karnal bunt diagnostic currently requires cumbersome sample treatments prior to morphological identification or DNA extraction, that are unsuitable for large-scale surveillance. Karnal bunt can be difficult to distinguish from ryegrass bunt (T. walkeri) and other smut and bunt fungi present in Australia using morphology and existing quantitative PCR (qPCR) assays lack specificity. This project aims to develop a pathway for both grain and air surveillance of Karnal Bunt to enable early detection in the event of an incursion, increasing the chance of successful containment and eradication. It will also improve Australia’s preparedness for a Karnal Bunt incursion by enabling seamless transition from first detection to effective surveillance through high throughput capability. Here, we report our work to 1) identify appropriate sampling locations and sample types throughout the grain handling pathway, 2) enrich the target in grain samples prior to DNA extraction and 3) provide sensitive diagnostic tests for triage and high-throughput requirements. Representative samples of each storage partition collected by the silos and named partition samples have been identified for high throughput grain surveillance, while samples for air surveillance will be best collected at grain receival points outside the grain storage facility. In addition, the dust fraction, collected by the silo extraction system has been identified as a low-resolution surveillance opportunity. Vacuum-based and mechanical-based prototypes were designed and evaluated for their efficiency to separate and concentrate the spores from the grain, while also considering cross contamination risks and cleaning options of the devices. The vacuum-based separation prototype provided higher spore recovery but presented more decontamination challenges compared to mechanical-based system. Various published and new qPCR assays targeting regions of the Karnal bunt genomes in either single or multiple copies have been evaluated. While assays targeting multi-copy genes demonstrated higher sensitivity, they exhibited lower specificity than single copy gene assays. Further investigations will focus on improving spore separation efficiency and decontamination of the high-throughput sample processing prototypes, developing fast DNA extraction methods from both grain and air samples to support at silo diagnostic, and improving new qPCR test specificity. Ultimately, the project will deliver enhanced Karnal Bunt surveillance system, from sampling to diagnostics, to aid the efficient implementation of both background and reactive surveillance.