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We analyzed lymphocyte subpopulations and cytokines 3 months after allogeneic hematopoietic stem cell transplantation aiming to identify predictive cellular and serum markers for chronic graft-versus-host disease (cGVHD). Samples of 49 patients (pts) (no cGVHD (n = 14), subsequent quiescent onset (n = 16), de novo onset of cGVHD (n = 19)) were analyzed in the absence of active GVHD by flow cytometry and enzyme-linked immunosorbent assay. All mean absolute cell counts are presented as cells per microliter; relative cell counts are presented as percentage of lymphocytes. Pts with subsequent de novo cGVHD had significantly higher relative and absolute counts of CD4+ T cells including higher absolute counts of CD4+ memory T cells (22.36%; 206.55/μl; 136/μl, respectively) compared to pts with subsequent quiescent onset of cGVHD (12.41%; 83.42/μl; 54.3/μl) and pts without cGVHD (10.55%) with regard to relative counts of CD4+ T cells. Similarly, significantly more relative and absolute regulatory T cell numbers (CD4+FOXP3+) were detected in pts with de novo onset of cGVHD (3.08% and 24.63/μl) compared to those in pts without (1.25% and 9.06/μl) or with quiescent onset of cGVHD (1.15% and 6.91/μl). Finally, relative B cell counts, including naïve and memory B cells, were also significantly decreased in pts developing quiescent cGVHD (0.85, 0.73, 0.12% resp.) when compared to pts with de novo onset (5.61, 5.24, 0.38%). The results demonstrate that alterations in immune reconstitution are already present before onset of clinical symptoms and differ between de novo and quiescent onset of disease.