Summary Penicilliosis caused by Talaromyces marneffei is a common AIDS ‐defining illness in South and Southeast Asia. Diagnosis is based on culture which can take up to 14 days for identification, leading to treatment delay and increased mortality. We developed a TaqMan real‐time PCR assay targeting the MP 1 gene encoding an abundant cell wall protein specific to T. marneffei . The assay's performance was evaluated in MP 1 ‐containing plasmids, clinical isolates, and plasma from HIV ‐infected patients with and without penicilliosis. The assay consistently detected 10 copies of MP 1 ‐containing plasmids per reaction and 100 T. marneffei yeast cells per millilitre plasma. There were no amplification with seven other Penicillium species and six other HIV ‐associated fungal pathogens tested. The assay was evaluated in 70 patients with AIDS : 50 patients with culture‐confirmed penicilliosis and 20 patients with opportunistic infections other than penicilliosis. The diagnostic sensitivity was 70.4% (19/27, 95% CI : 51.5–84.1%) and 52.2% (12/23, 95% CI : 33.0–70.8%) in plasma samples collected prior to and within 48 h of antifungal therapy respectively. The diagnostic specificity was 100% (20/20, 95% CI : 83.9–100%). This assay provides a useful tool for the rapid diagnosis of T. marneffei infection and has the potential to improve the management of patients with penicilliosis.