The continued emergence of human illness caused by avian influenza viruses (AIVs) demonstrates the threat of strains such as H5N1, H7N9, H10N8 and now H10N3. The genetic and biological properties of H10N3 viruses are not fully understood. In this study, three H10N3 strains isolated from live poultry markets (LPMs) were systematically studied. Genome sequencing showed that the poultry-origin viruses are highly homologous to the human H10N3 isolate. The three avian strains were A/chicken/Jiangsu/0146/2021(abbreviated as JS146, H10N3), A/chicken/Jiangsu/0169/2021 (JS169, H10N3) and A/chicken/Jiangsu/0189/2021(JS189, H10N3). Animal studies indicated that all three viruses are highly pathogenic to mice, and that all could replicate efficiently in mouse nasal turbinate and lungs despite maintaining their avian receptor binding affinity. We also found that these viruses replicated efficiently in A549 cells and chicken embryos. The strain JS146 had sensitivity to the neuraminidase-targeting drugs oseltamivir and zanamivir, whereas JS169 and JS189 were more resistant; genetic comparison implied that a substitution at NA position 368 conferred drug resistance. Importantly, several key molecular markers associated with mammalian adaptation had been detected in both avian and human-isolated H10N3 influenza viruses in the HA (G228S), PB2 (I292V and A588V), PB1 (M317V and I368V) and PA (A343S, K356R and S409N) protein. The above work contributes new insight into the biology of this potentially zoonotic subtype and provides evidence supporting the continued epidemiological monitoring of human infections caused by AIV subtype H10N3.