BACKGROUND: Little is known about viral co-infections in African human immunodeficiency virus (HIV)-infected children. We examined the prevalence of seromarkers for cytomegalovirus (CMV), herpes simplex virus type 2 (HSV-2) and hepatitis B virus (HBV) infections among HIV-infected, antiretroviral treatment (ART)-naive children in Lilongwe, Malawi. METHODS: Ninety-one serum samples were tested for IgG and IgM antibodies to CMV, and IgG antibodies to HSV-2 and hepatitis B surface antigen (HBsAg). Baseline demographic, clinical and laboratory data were abstracted from electronic records. RESULTS: CMV IgG was the most common positive result in all age groups (in 73% of children <1 year, and 100% in all other groups). Three patients were CMV IgM positive (3.3%), suggesting acute infection. HSV-2 IgG was positive in four patients (4.4%), and HBsAg in two (2.2%). CONCLUSIONS: CMV infection occurred early in life, and few children had specific signs of CMV infection at the time of ART initiation. Unrecognized HBV infection represents opportunities for testing and treatment of HIV/HBV co-infected children.