Background <p>The association between lung microbiome and pulmonary hypertension (PH) remain unknown. This study aims to define the airway mycobiome signature and its potential correlation with clinical parameters of PH.</p> Methods <p>Overall, 244 patients with PH and 120 healthy controls (CON) were recruited from three independent centers. The PH group was divided into subgroups not using antibiotics or corticosteroids (non-ANT/CORT), and those using ANT, CORT, or ANT + CORT within 1&#xa0;month, and clinical classification (Groups 1, 3, and 4), World Health Organization functional class (I–IV), and disease severity based on mean pulmonary artery pressure or pulmonary vascular resistance levels for in-depth comparison.</p> Results <p>Distinct airway mycobiome profiles were observed in PH, CON, and PH subgroups. Linear discriminant analysis effect size analysis showed increased <i>Purpureocillium</i>, <i>Issatchenkia</i>, and <i>Cyberlindnera</i> and decreased <i>Peroneutypa</i>, <i>Simplicillium</i>, and <i>Metarhizium</i> in patients with PH (non-ANT/CORT, ANT, CORT, and ANT + CORT) than in CON. Receiver operating characteristic analysis indicated a strong prediction of the two fungal genera sets in distinguishing PH and its subgroups from CON. The two major fungal phyla, <i>Ascomycota</i> and <i>Basidiomycota</i>, correlated differently with major clinical factors. Increased connections among the top fungal phyla or genera were observed in the PH than in the CON group. Dominant enrichment (<i>Purpureocillium</i>, <i>Issatchenkia</i>, and <i>Cyberlindnera</i>) and diminishment (<i>Peroneutypa</i>, <i>Simplicillium</i>, and <i>Metarhizium</i>) of fungal genera consistently and strongly predicted PH without being influenced by different PH subgroups.</p> Conclusions <p>This study provides the first description of the unique airway mycobiome signature in PH and among different PH subgroups.</p>