Abstract: This study is aimed to explore the value of metagenomic next-generation sequencing (mNGS) in diagnosing pathogen in fever patients. It is often a challenge to identify the pathogen that caused the infection in the HIV patients with fever. How could the mNGS be helpful for pathogen diagnosis is unclear. Here we reported a case of human immunodeficiency virus (HIV) patient with 2-month period of fever. After routine clinical laboratory tests including the conventional smear, culture, serological tests and pathological examinations, the causal pathogen still remained undiagnosed. Then the lymph node biopsy tissue was subjected to broad-range polymerase chain reaction (PCR) and the peripheral blood was subjected to mNGS. At the same time, peripheral blood culture was carried out with an extension of culture time to acquire the pathogen. Results from both broad-range PCR and mNGS revealed the pathogen was Talaromyces marneffei. The isolate recovered from the peripheral blood culture was subjected to the whole-genome sequencing. Whole genome sequencing revealed that the antimicrobial resistance gene FLU1 existed in this pathogen’s genome, but mNGS did not detect the FLU1 gene. Phylogenetic analysis based on whole genome sequence revealed that this isolate was far from other clones published in NCBI database. Here we reported a case of Talaromyces marneffei infection diagnosed by mNGS, showing that mNGS is helpful in etiological diagnosis for HIV patients with unexplained fever. However, application of mNGS in antimicrobial resistant genes detection and pathogen tracing need to be well-studied in the future.