Outbreaks of highly pathogenic avian influenza virus (HPAIV) may result in the infection of millions of poultry, causing devastating disease and up to 100% mortality. Avian influenza outbreaks and laboratory experiments have shown that HPAIV can emerge from low pathogenicity avian influenza virus (LPAIV) precursors. The multi-basic cleavage site (MBCS) in the haemagglutinin protein is described as the main pathogenic determinant of HPAIV infection in poultry. Identifying a precursor LPAIV is important for understanding the molecular changes involved in the emergence of HPAIV. In 2008, H7N7 HPAIV was confirmed in Oxfordshire, UK. The presence of a LPAIV precursor with a rare di-basic cleavage site (DBCS) was identified. The DBCS contains an additional basic amino acid compared to common circulating LPAIVs that harbour a single basic amino acid at the cleavage site (SBCS). Using reverse genetics, isogenic viruses based on A/chicken/England/11406/2008 H7N7 HPAIV, from the outbreak, were rescued with the MBCS replaced with either a DBCS (H7N7DB) as seen in the putative LPAIV precursor or a SBCS representative of common H7 LPAIVs (H7N7SB). Intravenous pathogenicity index testing of the recombinant viruses confirmed that only the MBCS conferred the highly pathogenic phenotype. Following passage in ovo, H7N7DB showed evidence of spontaneous evolution to a HPAIV genotype and phenotype as demonstrated by the acquisition of a MBCS, and by influenza virus-specific immunohistochemistry staining in embryo vascular endothelial cells. In contrast, deep sequencing of tissues from embryos in which H7N7SB was serially passaged up to three times showed retention of the LPAIV genotype. Thus, in chicken embryos, an H7N7 virus containing a DBCS displays an unstable nature allowing for rapid evolution to HPAIV. In ovo passage presents a novel approach to assess the likelihood of a LPAIV to evolve into HPAIV, and allows a laboratory-based dissection of molecular mechanisms behind the emergence of HPAIV.