Respiratory syncytial virus (RSV) may cause severe lower respiratory tract disease in premature infants. Prolonged viral shedding has been reported in patients with underlying immunosuppressive disorders, such as human immunodeficiency virus 1 (HIV-1) infection. During March to May 2006, 23 preterm pediatric patients developed nosocomial pneumonia in a district hospital in the Gauteng Province of South Africa due to RSV infection. The patients were identified using routine diagnostic testing. All had been admitted with their mothers to a Kangaroo Mother Care (KMC) ward from birth--a low care unit for the management of stable low birth weight infants. The HIV-1 seroprevalence among the mothers to these infants was 52.6%, translating to a 52.6% perinatal exposure. A multiplex nested RT-PCR was used to subtype RSV positive nasopharyngeal aspirates. Sequencing and phylogenetic analysis of part of the G-protein gene was used for molecular epidemiological analysis of the outbreak. In total, 19 of the 23 RSV positive specimens could be PCR amplified and sequenced. The subtype A, GA5 genotype was identified in 14 specimens and the BA genotype, a new subtype B genotype not previously recognized in South Africa, in seven. One patient had an infection with both genotypes. Phylogenetic analysis demonstrated eight separate introductions. Two of the strains identified in this outbreak were identical to strains circulating in a general pediatric ward of this hospital during the preceding month. Inadequate infection control measures by health care providers and mothers to children in KMC units may increase potentially the risk of severe RSV infection in a population group with compounded risk factors.