Avian influenza H9N2 viruses cause economic losses to the poultry industry and pose a public health risk. Two major H9N2 lineages dominate globally: the G1 lineage (genotype G1-B), prevalent in the Middle East, Africa and South Asia, and the BJ/94 lineage (predominantly genotype G57), dominant in China, Vietnam, South Korea, Indonesia and the Far East. We investigated replication, transmission and pathogenicity of prototype strains from these two lineages to link genotype to phenotype. The G57 virus A/Ck/Vietnam/H7F-14-BN4-315/2014 (Vietnam/315) was more lethal and showed greater tissue dissemination in chicken embryos than the G1-B virus A/chicken/Pakistan/UDL-01/2008 (Pakistan/UDL-01). Vietnam/315 exhibited higher replication in directly infected and contact chickens, with increased oropharyngeal and cloacal shedding and tissue dissemination. In contrast, the Pakistan/UDL-01 virus was shed mainly from the oropharynx at lower levels and remained localized to nasal tissues and trachea, highlighting differences in replication, tissue tropism and transmission. Gene analysis showed that the matrix (M) gene of Vietnam/315 enhanced replication in primary chicken kidney cells, while polymerase basic 2 (PB2), haemagglutinin (HA), neuraminidase (NA) and M genes promoted increased replication in Madin-Darby canine kidney cells. Both viruses preferentially bound to sulphated avian-like receptors. However, Vietnam/315 showed higher NA activity and a more acid-stable HA (pH fusion 5.2) than Pakistan/UDL-01. These findings indicate that the G57 genotype strain examined exhibits greater replication and transmission fitness than the G1-B lineage strain in vivo, ex vivo and in vitro. Although based on prototype viruses, the results suggest that reassortment involving G57 genotype genes could enhance viral fitness and increase animal and human infection risk.