Purpose: To identify diagnostic and prognostic markers of chronic graft-versus-host disease (cGVHD), the major cause of morbidity and mortality after allogeneic hematopoietic cell transplantation (HCT). Patients and Methods: Using a quantitative proteomics approach, we compared pooled plasma samples obtained at matched time points after HCT (median, 103 days) from 35 patients with cGVHD and 18 without cGVHD (data are available via ProteomeXchange with identifier PXD002762). Of 105 proteins showing at least a 1.25-fold difference in expression, 22 were selected on the basis of involvement in relevant pathways and enzyme-linked immunosorbent assay availability. Chemokine (C-X-C motif) ligand 9 (CXCL9) and suppression of tumorigenicity 2 (ST2) also were measured on the basis of previously determined associations with GVHD. Concentrations of the four lead biomarkers were measured at or after diagnosis in plasma from two independent verification cohorts (n = 391) to determine their association with cGVHD. Their prognostic ability when measured at approximately day +100 after HCT was evaluated in plasma of a second verification cohort (n = 172). Results: Of 24 proteins measured in the first verification cohort, nine proteins were associated with cGVHD, and only four (ST2, CXCL9, matrix metalloproteinase 3, and osteopontin) were necessary to compose a four-biomarker panel with an area under the receiver operating characteristic curve (AUC) of 0.89 and significant correlation with cGVHD diagnosis, cGVHD severity, and nonrelapse mortality. In a second verification cohort, this panel distinguished patients with cGVHD (AUC, 0.75), and finally, the panel measured at day +100 could predict cGVHD occurring within the next 3 months with an AUC of 0.67 and 0.79 without and with known clinical risk factors, respectively. Conclusion: We conclude that the biomarker panel measured at diagnosis or day +100 after HCT may allow patient stratification according to risk of cGVHD.