Abstract： Objective To explore the clinical features of cytomegalovirus (CMV) and EpsteinBarr virus (EBV) infection after second hematopoietic stem cell transplantation (HSCT).Methods Twenty-five patients after second HSCT from Sep.2009 to Oct.2016 were collected,and CMV and EBV DNA in peripheral blood was detected regularly by polymerase chain reaction (PCR).Factors associated were compared by univariate analysis.Results The total incidence of CMV infection was 52.0％ (13/25) after second HSCT.The incidence of CMV infection was 100％ (2/2),33.3％ (5/15) and 75％ (6/8) in bone marrow group,peripheral blood stem cell group,and mixed group,respectively.Stem cell sources were significantly correlated with CMV infection (P =0.038),however,there was no significant difference in CMV infection rate among three groups (P＞0.05).None of preconditioning regimen,GVHD prophylaxis programs or severity of aGVHD were correlated with CMV infection after second HSCT (P＞0.05).The total incidence of EBV infection was 24.0％ (6/25) after second HSCT.The incidence of EBV infection was 100％ (2/2),6.7％ (1/15) and 37.5％ (3/8) in bone marrow group,peripheral blood stem cell group,and mixed group,respectively.Stem cell sources were significantly correlated with EBV infection (P =0.008).The EBV infection rate in bone marrow group was significantly higher than that in peripheral blood group (P =0.022),however,no significant differences were found between bone marrow group and mixed group,as well as between peripheral blood group and mixed group (P＞0.05).Transplant methods were significantly correlated with EBV infection (P =0.007).The EBV infection rate in haploidentical HSCT group (71.4％) was significantly higher than that in HLA-matched sibling HSCT group (0％) and autologous HSCT group (0％) (P =0.021 and 0.028),however,no significant differences were found between any other two groups (P＞0.05).None of preconditioning regimen,GVHD prophylaxis programs or severity of aGVHD were correlated with EBV infection after second HSCT (P＞0.05).Conclusion The incidence of CMV and EBV infection in patients undergoing second HSCT is high.Stem cell sources and transplant methods are associated with CMV and EBV infection after second HSCT.