Microbial translocation (MT) from the gut is implicated in driving immune activation, increasing morbidity and mortality in HIV. We used bacterial 16S rDNA PCR, Sanger sequencing, and high-throughput sequencing to identify microbial DNA in the bloodstream of HIV-infected children in London, United Kingdom. Blood samples were collected from sequential children attending the HIV clinic at Great Ormond Street Hospital, London. DNA extraction, broad range 16S rDNA PCR, and standard Sanger sequencing were carried out. A subset of positive samples was analyzed by high-throughput sequencing (Roche 454 platform). Of 105 samples collected from sequential children, nine were positive using broad range 16S rDNA PCR (8.6%; 95% CI 4.4–16%). From three amplicons, 16S rDNA sequences were identified as Streptococcus, Propionibacterium acnes, and coagulase-negative Staphylococcus. Four positive samples were analyzed by high-throughput sequencing. In the three samples in which organisms were identified by Sanger sequencing, the same species were identified. Further species, in differing proportions, were identified in all four samples. The identified organisms included known gut orders Bifidobacteriaceae, Lactobacillaceae, Bacteroidales, and Clostridiales. In immunocompetent children of equivalent age, no bacterial DNA was detected in blood using this approach. This is the first study to our knowledge using molecular techniques to identify MT in children in the developed world. Our data indicate that 16S rDNA is detectable in 8.6% of HIV-infected children. Levels of DNA were low and from multiple bacterial species. Further studies are needed to ascertain the importance of MT in HIV-infected children.