Human Cytomegalovirus (HCMV) is one of the most important and critical viral cause of graft rejection and threatening of surveillance in Bone marrow Transplant (BMT) recipients. Active and latent HCMV infections are widespread in more than 90% of BMT donors and recipients and monitoring of this viral infection has a critical role in management of BMT clinical complications. In this research, two new sensitive and specific nested-PCR protocols were optimized for accurate detection of HCMV infection in BMT donors and recipients. Pre and post transplantation also the prevalence of HCMV-UL55 genotypes were detected in these patients. In this cohort and retrospective study blood (plasma and Buffy coat) and urine samples were collected for 6 years from 110 BMT patients pre-transplantation and followed for 100 days post-transplantation. Also, blood and urine samples were simultaneously collected from 98 BMT donors. HCMV-UL55-nested-PCR and HCMV-UL4-nested-PCR methods were optimized and used for detection of HCMV infection in all collected samples from transplant donors and recipients. The genotyping of HCMV UL55 gene were done for all UL55-nested PCR positive samples. BMT donors and recipient’s laboratory and clinical data were collected and statistically analyzed with the version 15 of SPSS soft ware. UL4-nested-PCR positive results were detected in plasma 3045/4950 (61.5 %), Buffy coat 3168/4950 (64%) and urine 3020/4950 (61 %) samples of BMT recipients post-transplantation. Also, UL55-nested-PCR positive results were diagnosed in 3540/4950 (71.5 %), 3634/4950 (73.4 %) and 3292/4950 (66.5 %) of these samples respectively. Mean of 25% of transplant donors were infected totally with HCMV infection. The decline pattern of the prevalence of UL55 genotypes in plasma, Buffy coat and Urine samples were as followed: gB2>gB3> gB1> gB4, gB2> gB1> gB3> gB4 and gB2> gB3> gB1> gB4 In conclusion, Increasing of HCMV infection when switching from pre to post BMT conditions and detection of gB2 UL55 genotype in the most samples of BMT patients and also reviewing the result of another HCMV researchers announced the need of completed investigators focused on the study of the possible relationships between the HCMV UL55 genotype and the impact of HCMV infection in BMT recipients.