A nosocomial Hepatitis B virus (HBV) outbreak at a paediatric onco-haematology unit was investigated using molecular biological methods to determine the origin of the infections. The National Reference Laboratory of Hepatitis Viruses received seven HBsAg positive sera from patients and one from the brother of a patient. A fragment of the preS1/preS2/S genes from all samples was amplified, the PCR products were sequenced and a rooted phylogenetic tree was constructed. All nucleotide sequences from the different patients were very similar and 6 of the 8 sequences were identical, suggesting a common origin of the infections. These sequences were closely related to those amplified from a nosocomial HBV epidemic in another hospital in Hungary. The on-scene investigation revealed several malpractices. The two hospital departments had close connections and some of the patients were treated in both institutions. Present report underlines the importance of developing screening protocols for hepatitis viruses and that of the introduction of regular training programs for health care professionals in the field of hospital hygiene.