Hematopoietic chimerism was monitored in 18 patients with various diseases after gender-mismatched allogeneic stem cell transplantation (alloSCT). To detect host and donor cells, FISH analysis of sex chromosomes was applied. X and Y chromosomes were detected simultaneously in interphase nuclei by two-color probes. Chimerism was examined sequentially in post-transplant peripheral blood and bone marrow as well as in purified T cells. Patients with complete donor or decreasing host chimerism have not rejected or relapsed but experienced a high incidence of acute graft versus host disease (aGvHD). The clinical value of stable mixed chimerism detection remains uncertain. However, it appears to be associated with a lower risk of aGvHD. Three patients with an increase in host cells rejected their grafts. The immunotherapy was introduced to four other patients with increasing host chimerism. All of them responded, however, one relapsed in CNS despite the conversion to complete donor chimerism in both bone marrow and peripheral blood. We concluded that two-color FISH analysis of sex chromosomes was a valuable tool for chimerism monitoring and provided significant clinical data.