Clinical diagnosis of sepsis in newborn infants is not easy and there is no laboratory test with 100% specificity and sensitivity, with the exception of blood culture, the results of which are not available for at least 48–72 h. Polymerase chain reaction methodology has been used to diagnose different bacterial, viral and protozoal infections, and the possibility of amplifying the DNA region common to all bacteria could represent an optimal method for the diagnosis of sepsis. The authors have performed PCR in a group of 33 neonates at risk for early-onset sepsis, correlating molecular data with blood culture results. The presence of bacterial DNA in blood samples was evaluated, amplifying the DNA region encoding the 16S rRNA. There were no false negative results (four positive blood cultures and four positive PCR), with competitive costs and time. This method also allows the diagnosis of sepsis due to uncommon species and also, using a second PCR with specific primers, an aetiological diagnosis.