Hematopoietic chimerism (HpC) was assayed monthly using a sensitive, polymerase chain reaction (PCR) -based method in consecutive patients. Between January 1998 and April 2002, 181 patients underwent non-T cell depleted allogeneic hematopoietic cell transplantation (HCT). A total of 163 patients were evaluable for HpC at 1 month (11 early deaths; no informative band for HpC analysis/no genomic DNA in seven). In all, 53 of 163 patients (33%, median recipient DNA of 15% (range 5-95)), 39 of 151 patients (26%), and 27 of 142 patients (19%) showed mixed chimerism (MC) at 1, 2, and 3 months after HCT, respectively. Conditioning regimen (busulfan-fludarabine-ATG vs BuCy, relative risk 3.99 (95% CI 1.16-10.92)), neutrophil engraftment (>/=day 17 vs </=day 16, relative risk 2.49 (95% CI 1.14-5.41)), and acute graft-versus-host disease within 30 days (none vs yes, relative risk 4.78 (95% CI 1.50-15.17)) were independent variables that showed significant correlation with having >/=5% recipient DNA at 1 month. Five patients experienced secondary graft failure. All five patients showed MC at 1 month with median recipient DNA of 40%. None of the 109 patients with complete chimerism experienced graft failure (P=0.002). Our study showed that MC shown on monthly analysis of HpC after allogeneic HCT is a significant predictor of secondary graft failure. Bone Marrow Transplantation (2003) 32, 423-431. doi:10.1038/sj.bmt.1704147