Imbalances of iron homeostasis are associated with an adverse clinical outcome of pulmonary hypertension (PH). Herein, we aimed to analyze the impact of iron deficiency (ID) in a real-life PH patient cohort according to different currently used ID definitions. In a retrospective study including 153 precapillary PH patients followed over a mean period of five years, iron deficiency was assessed according to five clinical definitions used in previous trials. The impact of ID on clinical, hematological and hemodynamic parameters was investigated. Depending on the different cutoff levels for serum ferritin and transferrin saturation, currently used ID definitions indicated a prevalence of either true or functional ID in 11 to 75 percent of PH patients. A good diagnostic accuracy was achieved by using the sTFRF/log ferritin (sTFRF) index, which identified 33 to 42 percent of PH patients as being iron deficient. The sTFRF index had the best prediction for the association between ID and clinical outcome. Iron deficient patients with precapillary PH had a significantly higher mortality as compared to non-iron deficiency subjects, which was true for both, PH patients with and without anemia. Although levels of the iron hormone hepcidin were rather affected by ID than by inflammation, they were not associated with the clinical course or mortality of PH subjects. To conclude, ID had a significant impact on the clinical course of precapillary PH patients. The appropriate use of robust biomarkers to define ID is a prerequisite to further evaluate the role of ID and the potential benefit of iron supplementation in precapillary PH patients.