BACKGROUND Ramularia leaf spot (RLS), caused by Ramularia collo‐cygni, is an emerging threat to barley (Hordeum vulgare L.) production. RLS has been reported in Australia, however only minimal information is available regarding its detection and distribution. Due to initial asymptomatic growth in planta, slow growth in vitro and symptomatic similarities to net blotch and physiological leaf spots, detection of this pathogen can be challenging. Quantitative polymerase chain reaction (PCR)‐based methods for R. collo‐cygni‐specific identification and detection have been described, however these assays have been demonstrated to lack specificity. False‐positive detections may have serious implications, thus we aimed to design a robust R. collo‐cygni‐specific PCR method. RESULTS Using the phylogenetically informative RNA polymerase II second largest subunit (rpb2) and translation elongation factor 1‐alpha (tef1‐α) genes, along with the tef1‐α gene of H. vulgare, a triplex assay was developed for both quantitative and droplet digital PCR. The triplex assay detected R. collo‐cygni DNA in barley leaves from New South Wales, South Australia, Tasmania, Victoria and Western Australia. No R. collo‐cygni DNA was detected in barley seed grown in Western Australia. CONCLUSION The presence of R. collo‐cygni DNA has been confirmed in Australian barley crops, suggesting a distribution ranging across the southern barley growing regions of Australia. The R. collo‐cygni‐specific assay will be a valuable tool to assist with monitoring the distribution and impact of R. collo‐cygni in Australia and other regions. © 2021 Society of Chemical Industry. Quantitative and droplet digital polymerase chain reaction (PCR) confirms presence of Ramularia collo‐cygni in southern barley growing regions of Australia.